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WO2024163762A2 - Six membered ring containing oligomers - Google Patents

Six membered ring containing oligomers Download PDF

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Publication number
WO2024163762A2
WO2024163762A2 PCT/US2024/014036 US2024014036W WO2024163762A2 WO 2024163762 A2 WO2024163762 A2 WO 2024163762A2 US 2024014036 W US2024014036 W US 2024014036W WO 2024163762 A2 WO2024163762 A2 WO 2024163762A2
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WO
WIPO (PCT)
Prior art keywords
nucleotide
antisense strand
formula
oligonucleotide
counting
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PCT/US2024/014036
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French (fr)
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WO2024163762A3 (en
Inventor
Muthiah Manoharan
Masaaki AKABANE-NAKATA
Dhrubajyoti Datta
Jayanta KUNDU
Original Assignee
Alnylam Pharmaceuticals, Inc.
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Application filed by Alnylam Pharmaceuticals, Inc. filed Critical Alnylam Pharmaceuticals, Inc.
Publication of WO2024163762A2 publication Critical patent/WO2024163762A2/en
Publication of WO2024163762A3 publication Critical patent/WO2024163762A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07F9/6533—Six-membered rings

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. For example, 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

The present disclosure relates generally to six-membered ring containing nucleosides particularly, piperidino nucleosides and oligonucleotides, oligomers derived from the six-membered ring monomers comprising the same.

Description

SIX MEMBERED RING CONTAINING OLIGOMERS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/442,570, filed February 1, 2023, contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 31, 2024, is named “051058-000102WOPT SEQ_list.xml” and is 2,740,425 bytes in size. TECHNICAL FIELD [0003] The present disclosure relates generally to six-membered nucleosides and oligonucleotides and oligomers comprising the same. BACKGROUND [0004] 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. The chemical synthesis of PMOs has been conventionally carried out using N-tritylated 5’- chlorophospho-ramidate morpholino monomers which have poor solution-phase stability, long condensation times and low stepwise condensation yields, making PMO synthesis relatively expensive and inaccessible. See, for example, Krishna et al., FEBS Lett. (2019), vol 593(13), pp. 1459-1467. The methods and monomers described herein don’t have these limitations. The methods and monomers described herein allow synthesis of PMOs and similar structures at scale and relatively inexpensively. [0006] In one aspect, provided herein is an oligonucleotide comprising at least one nucleoside of Formula (IV) (e.g., one):
Figure imgf000004_0001
Formula (IV)
[0007] In nucleotides of Fomrula (IV), B’ is an optionally modified nucleobase.
[0008] In nucleotides of Formula (IV), XM can be CH2, O, NRN or S, where RN 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. For example, XM is CH2, O or NH. In some embodiments, XM is CH2. In some embodiments, XM is O. In yet some other embedments, XM is S.
[0009] In nucleotides of Formula (IV), R43 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, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or -0-C4-3oalkyl- ON(CH2R8)(CH2R9). In some embodiments of any one the aspects described herein, R43 is a bond to an intemucleotide linkage to a subsequent nucleotide. In some other embodiments of any one of the aspects described herein, R43 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, R43 is H. In still some other embodiments of any one of the aspects described herein, R43 is a nitrogen protecting group, e.g., triphenylmethyl (trityl). In yet some other embodiments of any one of the aspects described herein, R43 is hydroxyl or a protected hydroxyl. In yet some other embodiments of any one of the aspects described herein, R43 is hydroxyl. In yet some other embodiments of any one of the aspects described herein, R43 is a protected hydroxyl.
[0010] In nucleotides of Formula (IV), R45 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, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O- C4-3oalkyl-ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P- 0-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((H0)2(0)P-0-(H0)(0)P-0- P(H0)(0)-0-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha- thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((H0)2(0)P- NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates [(Rp)(0H)(0)P-0-5', Rp is optionally substituted Ci-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(Rpl)(0H)(0)P-0- 5', RP1 is alkoxyalkyl, e.g., methoxymethyl (CH20Me) or ethoxymethyl ], (HO)2(X)P-O[-(CH2)a- 0-P(X)(0H)-0]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O- P(X)(0H)-0]b- 5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-0- P(X)(0H)-0]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H0[-(CH2)a-P(X)(0H)-0]b- 5' , H2N[- (CH2)a-P(X)(0H)-0]b- 5', H[-(CH2)a-P(X)(0H)-0]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group (e.g., - CH=CH-XP, Xp is a phosphonate group) or C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), wherein: X is O or S;a and b are each independently 1-10; and each R8 and R9 is independently H, a targeting ligand (e.g., N-Acetylgalactosamine (GalNAc)), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl.
[0011] In some embodiments, R45 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 R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group. For example, R45 is hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate, or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group. In some embodiments, R45 is a bond to an intemucleotide linkage to a preceding nucleotide. In some embodiments, R45 is hydroxyl or protected hydroxyl.
[0012] It is noted only one of R43 and R45 can be a solid support or a linker covalently bonded to a solid support. [0013] In some embodiments, 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. For example, when the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, R45 is a bond to a phosphodiester intemucleotide linkage. In another example, when the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, R45 is a bond to a phosphorothioate intemucleotide linkage.
[0014] In some embodiments of any one of the aspects described herein, an oligonucleotide described herein comprises two or more consective nucleosides of Formula (IV). For example, the oligonucleotide comprises:
Figure imgf000006_0001
where: nD 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);
XD is O or S; each RP2 is independently optionally substituted Ci-ealkyl (e.g., methyl); and
XM, B’, R43 and R45 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.
[0015] In some embodiments of any one of the aspects described herein, nD is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. For example, nD is 1, 2, 3, 4, 5 or 6. In some embodiments of any one of the aspects described herien, nD is 1, 2, 3 or 4. For example, nD is 1 or 2.
[0016] In some embodiments of any one of the aspects described herien, XD is O.
[0017] In some oligonucleotides described herein and comprising two or more nucleosides of Formula (IV), all XM are same. For example, all XM are CH2. In some other examples, all XM are O. In some other examples, all XM are S. In some other examples, at least one XM is not O. [0018] In some embodiments of any one of the aspects described herein, the oliognucleotides is 4 nucleotides in length, nD is 4, and the oligonucleotide does no comprise the sequence 5’-UCAG- 3’.
[0019] In some oligonucleotides described herein and comprising two or more nucleosides of Formula (IV), at least one of XM is CH2 and at least one XM is O.
[0020] In another aspect, provided herein is a compound of Formula (III):
Figure imgf000007_0001
(Formula III).
[0021] In compounds of Formula (III), B’ is an optionally modified nucleobase.
[0022] In compounds of Formula (III), XM is CH2, O, NRN or S, where RN 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. For example, XM can be CH2, O or NRN. In some embodiments of any one of the aspects described herein, XM is CH2, O or NH. For example, XM is CH2. In some cases XMis O. In some other non-limiting exampples, XM is S. [0023] In compounds of Formula (III), R33 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, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), or -0-C4-3oalkyl-ON(CH2R8)(CH2R9). In some embodiments of any one of the aspcets described herein, R33 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. For example, R33 is a reactive phosphorous group, a solid support, a linker, a linker covalently bonded to a solid support, or a nitrogen protecting group. In some embodiments of any one of the aspects described herein, R33 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. In some other embodiments of any one of the aspects described herein, R33 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, R33 is H. In still some other embodiments of any one of the aspects described herein, R33 is a nitrogen protecting group, e.g., truphenylmethyl (trityl).
[0024] In compounds of Formula (III), R35 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(CH2R8)(CH2R9), -0-C4-3oalkyl- ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)- 0-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P- 0-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates (R(0H)(0)P-0-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc...), alkyletherphosphonates (R(0H)(0)P-0-5', R=alkylether, e.g., methoxymethyl (CFbOMe), ethoxymethyl, etc...), (HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a- P(X)(0H)-0]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0- P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-0-P(X)(0H)-0]b- 5', Me2N[- (CH2)a-0-P(X)(0H)-0]b- 5', H0[-(CH2)a-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[- (CH2)a-P(X)(0H)-0]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate) group; wherein X is O or S; and a and b are each independently 1- 10); and each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl. In some embodiments of any one of the aspects described herein, R35 is hydroxy or protected hydroxy. For example, R35 is a protected hydroxyl (e.g., 4,4'-dimethoxytrityl- protected). In yet some other embodiments of any one of the aspects described herein, R35 is a phosphate group. In still some other embodiments of any one of the aspects described herein, R35 taken together with the carbon to which it is attached can form is a vinyl phosphonate group.
[0025] In compounds of Formula (III), R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group, a C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate) group. [0026] In some embodiments of any one of the aspects described herein, R35 is a reactive phosphorous group. For example, the R35 is -OP(O)(RP4)(N(RP2)2), where RP4 is Cl and each RP2 is methyl.
[0027] It is noted only one of R33 and R35 can be a reactive phosphorous group, a solid support, or a linker covalently bonded to a solid support.
[0028] In some embodiments of any one of the aspects describd herein, R33 is a solid support, or a linker covalently bonded to a solid support. For example, R33 is a solid support, or a linker covalently bonded to a solid support, and R35 is hydroxyl or protected hydroxyl group. In some compounds of Formula (III), XM is CH2, O, NRN or S; R33 is a solid support, or a linker covalently bonded to a solid support; and R35 is hydroxyl or protected hydroxyl group (e.g., dimethoxy trityl). [0029] In some embodiments of any one of the aspects described herein, R33 is a reactive phosphorous group. For example, the R33 is -OP(O)(RP4)(N(RP2)2), where RP4 is Cl and each RP2 is methyl. In some embodiments, R33 is a reactive phosphorous group and R35 is hydroxyl or protected hydroxyl group. For example, R33 is -OP(O)(RP4)(N(RP2)2), where RP4 is Cl and each RP2 is methyl; and R35 is hydroxyl or protected hydroxyl group (e.g., dimethoxytriryl).
[0030] 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).
[0031] In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at one of positions 2-9, counting from the 5 ’end of the oligonucleotide. For example, 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. In some embodiments, 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.
[0032] In some embodiments, the oligonucleotide described herein is double-stranded. For example, 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.
[0033] Accordingly, in another aspect, provided herein is 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.
[0034] In some embodiments, the double-stranded nucleic acid is a double-stranded RNA. For example, the double-stranded nucleic acid is an siRNA.
[0035] In some embodiments, 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). For example, 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). In some embodiments, 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). In one non-limiting example, 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). In another non-limiting example, 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). In yet another non-limiting example, 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). In still another non-limiting example, 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).
[0036] In some embodiments, 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). For example, 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).
[0037] In some embodiments, 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). In one non-limiting example, 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). In another non-limiting example, 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). In yet another non-limiting example, 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). In still another non-limiting example, 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).
[0038] In another aspect, provided herein is a method for inhibiting or reducing the expression of a target gene in a subject. The method 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.
[0039] It is noted that in oligonucleotides comprising a nucleotide of Formula (IV), at least one of R43 and R45 is a bond to a intemucleotide linkage. Thus, when R43 is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R45 is a bond to an intemucleotide linkage to a preceding nucleotide. Conversely, when R45 is not a bond to an intemucleotide linkage to a preceding nucleotide, then R43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing (s) will be provided by the Office upon request and payment of the necessary fee.
[0041] FIG. 1 depicts some exemplary compounds comprising C5 or C4 modified pyrimidines according to some embodiments of the disclosure.
[0042] FIG. 2 depicts some exemplary compounds comprising N2 modified purines according to some embodiments of the disclosure.
[0043] FIG. 3 depicts some exemplary compounds comprising N6 modified purines according to some embodiments of the disclosure.
[0044] FIG. 4 depicts some exemplary compounds comprising N7-deaza or C-8 modified purines according to some embodiments of the disclosure.
[0045] FIG. 5 depicts some exemplary modified car-morpholino, vinylphosphonate and CPG compounds comprising N2 modified purines according to some embodiments of the disclosure.
[0046] FIG. 6 depicts exemplary synthesis scheme for preparing C5-modified pyrimidine car- PMO compounds according to some embodiments of the disclosure.
[0047] FIG. 7 depicts some exemplary ligands.
[0048] 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).
[0049] FIG. 9 depicts crystal structures of selected intermediates.
[0050] 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.
[0051] FIGS. 11A and 11B are general schemes for synstheis of exemplary car-morpholino and morpholino amidites.
[0052] FIG. 12 are general synthesis schemes for car-morpholino-VP, morpholino-VP- amidite and CPG.
[0053] 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). [0054] FIG. 14 shows structures of some monomer abbreviations used in the nucleic acid sequences described herein.
[0055] FIG. 15A shows exemplary oligonucleotides comprising six membered ring monomers I-IV shown in FIG. 14.
[0056] FIG. 15B shows exemplary oligonucleotides comprising six membered ring monomers Y271-Y274 shown in FIG. 14.
[0057] 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).
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] FIG. 21 depicts a scheme showing 5’-5-morpholino-U monomer synthesis.
[0063] FIG. 22 depicts a scheme showing 5’-5-morpholino-C monomer synthesis.
[0064] FIG. 23 depicts a scheme showing 5’-5-morpholino-A monomer synthesis.
[0065] FIG. 24 depicts a scheme showing 5’-5-morpholino-G monomer synthesis.
[0066] FIG. 25 depicts a scheme showing 5’-5-morpholino (phosphorami dites) monomers synthesis and oligonucleotide ynthesis strategy.
[0067] FIG. 26 depicts a scheme showing 5’-5-morpholino (chlorophosphorami dates) monomers synthesis and oligonucleotide synthesis strategy. [0068] FIG. 27 depicts a scheme showing A-acetyl piperazino (chlorophosphoramidates) monomers synthesis.
[0069] FIG. 28 depicts a scheme showing A-acetyl piperazino (phosphoramidites) monomers synthesis.
[0070] FIG. 29 depicts a scheme showing A-acetyl piperazino (chlorophosphoramidates) monomers synthesis and oligonucleotide synthesis scheme.
[0071] FIG. 30 depicts a scheme showing N- acetyl piperazino (phosphoramidites) monomers synthesis and oligonucleotide synthesis scheme.
[0072] FIG. 31 depicts some exemplary modified PMO sequences. Seqeunces shown are, from top to bottom, SEQ ID NOs: 241-244.
DETAILED DESCRIPTION
[0073] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
[0074] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
Nucleobase
[0051] In some embodiments of the various aspects described herein, B’ is an optionally modified nucleobase. It is noted that the nucleobase can be a natural or non-natural nucleobase. By a “non-natural nucleobase” is meant a nucleobase other than adenine, guanine, cytosine, uracil, or thymine. Exemplary 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-substituted adenines and guanines, 5 -trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylguanine, 5 -substituted pyrimidines, 6- azapyrimi dines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5- alkyluracil (e.g., 5 -methyluracil), 5 -alkylcytosines (e.g., 5-methylcytosine), 7-alkylguanine, 5- alkyl cytosine, 7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3 -methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5 -nitroindole, 3 -nitropyrrole, 5- methoxyuracil, uracil-5-oxyacetic acid, 5 -methoxy carbonylmethyluracil, 5-methyl-2-thiouracil, 5- methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino- 3carboxypropyl)uracil, 3 -methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N- methylguanines, or O-alkylated bases. Further 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.
[0052] In some embodiments, 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,
2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8- (hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6, N6-(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6- (alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8- (hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine,
3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5- (alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil,5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)- 2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5- (allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(l,3- diazole-l-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4- (dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2- (thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)- 4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1 -substituted pseudouracil, 1 -substituted 2(thio)-pseudouracil, 1 -substituted 4-(thio)pseudouracil, 1 -substituted 2,4-(dithio)pseudouracil, 1 -(aminocarbonylethylenyl)-pseudouracil, 1 -(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1 -(aminocarbonyl ethylenyl)- 4-(thio)pseudouracil, l-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,
1 -(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1 -(aminoalkylamino-carbonylethylenyl)- 2(thio)-pseudouracil, 1 -(aminoalkylaminocarbonyl ethylenyl)-4-(thio)pseudouracil, l-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, l,3-(diaza)-2-(oxo)-phenoxazin- 1-yl, l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, l-(aza)-2- (thio)-3-(aza)-phenthiazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7- substituted l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7-(aminoalkylhy droxyl)-!, 3 -(diaza)-2- (oxo)-phenoxazin- 1 -yl, 7-(aminoalkylhy droxyl)- 1 -(aza)-2-(thio)-3 -(aza)-phenoxazin- 1 -yl, 7- (aminoalkylhydroxyl)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7-(aminoalkylhy droxyl)- l-(aza)-2- (thio)-3 -(aza)-phenthiazin- 1 -yl, 7-(guanidiniumalkylhy droxyl)- 1 ,3 -(diaza)-2-(oxo)-phenoxazin- 1 - yl, 7-(guanidiniumalkylhydroxyl)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-(guanidiniumalkyl- hy droxyl)- 1 ,3 -(diaza)-2-(oxo)-phenthiazin- 1 -yl, 7-(guanidiniumalkylhy droxyl)- 1 -(aza)-2-(thio)- 3-(aza)-phenthiazin-l-yl, l,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3- (methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7- (aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5- (trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6- (methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5 -nitroindole, 3 -nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N2-substituted purines, N6-substituted purines, (/-substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof. [0053] In some embodiments, 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. Examples of the 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, N2- and N6- 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.
[0054] In some embodiments of any one of the aspects, the non-natural nucleobase is a universal nucleobase. As used herein, 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, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof.
[0055] In some embodiments of any one of the aspects described herein, the non-matural nucleobase is a protected nucleobase. As used herein, a “protected nucleobase” referes to a nucleobase comprising a nitrogen protecting group, and/or an oxygen protecting group, and/or a sulfur protecting group.
[0056] In some embodiments of any one of the aspects described herein, the non-natural nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.
[0057] In some embodiments of any one of the aspects described herein, the nucleobase is a pyrimidine modified at the C4 position.
[0058] In some embodiments of any one of the aspects described herein, the nucleobase is a pyrimidine modified at the C5 position.
[0059] In some embodiments of any one of the aspects described herein, 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.
[0060] In some embodiments of any one of the aspects described herein, the nucleobase is a purine modified at the C6 position. [0061] In some embodiments of any one of the aspects described herein, the nucleobase is a N-7 deaza purine, optionally modified at the N7 position.
[0062] In some embodiments of any one of the aspects described herein, the nucleobase is selected from the group consisting
Figure imgf000018_0001
Figure imgf000018_0002
Figure imgf000019_0001
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.
[0063] In some embodiments of any one of the aspects described herein, XM can be CH2, O,
S, or NRN.
[0064] In some embodiments of any one of the aspects described herein, XM is CH2.
[0065] In some embodiments of any one of the aspects described herein, XM is O.
[0066] In some embodiments of any one of the aspects described herein, XM is S.
[0067] In some embodiments of any one of the aspects described herein, XM is NRN. For example, XM is NH.
R33
[0068] In some embodiments of any one of the aspects described herein, R33 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, dialkylamino, -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or -0-C4-3oalkyl- ON(CH2R8)(CH2R9).
[0069] In some embodiments of any one of the aspects described herein, R33 is a nitrogen protecting group. For example, R33' can be triphenylmethylamine (Tr), [(4- methoxyphenyl)diphenylmethyl]amine (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr) or trifluoroacetamide. In some embodiments of any one of the aspects described herein, R33 is triphenylmethylamine or trifluoroacetamide. For example, R33 is triphenylmethylamine. [0070] In some embodiments of any one of the aspects described herein, R33 is a reactive phosphorus group. For example, R33 is -OP(ORP)(N(RP2)2), -OP(SRP)(N(RP2)2), - OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(NRP2)2, -OP(O)(ORP)H, - OP(S)(ORP)H, -OP(O)(SRP)H, -OP(O)(ORP)RP3, -OP(S)(ORP)RP3, or -OP(O)(SRP)RP3. [0071] In some embodiments of any one of the aspects, R33 is -OP(ORP)(N(RP2)2), - OP(SRP)(N(RP2)2), -OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(N(RP2)2), - OP(O)(ORP)H, -OP(S)(ORP)H, where RP is an optionally substituted C1-6alkyl, each RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. [0072] In some embodiments of any one of the aspects, R33 is -OP(ORP)(N(RP2)2). For example, the R33 is -OP(ORP)(N(RP2)2), where RP is cyanoethyl (-CH2CH2CN) and each RP2 is isopropyl. [0073] In some embodiments of any one of the aspects descried herein, R33 is a solid support or a linker covalently attached to a solid support. For example, R33 is –OC(O)CH2CH2C(O)NH-Z, where Z is a solid support. [0074] In some embodiments of any one of the aspects described herein, R33 is - O(CH2CH2O)rCH2CH2OR334, where r can be 1-50; R334 is independently for each occurrence H, C1-C30alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R335; and R335 is independently for each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino. [0075] In some embodiments of any one of the aspects described herein, R33 is - (CH2CH2NH)sCH2CH2-R335, where s can be 1-50 and R335 can be independently for each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino. [0076] In some embodiments of any one of the aspects described herein, R33 is hydrogen. [0077] In some embodiments of any one of the aspects described herein, R33 is hydroxyl or protected hydroxyl. In some embodiments of any one of the aspects described herein, R33 is hydroxyl. In some embodiments of any one of the aspects described herein, R33 is protected hydroxyl. [0078] In some embodiments of any one of the aspects described herein, R33 is C1-C30alkoxy optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2— [CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R33 is C1-C30alkoxy optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy. In some embodiments of any one of the aspects described herein, R33 is –O(CH2)tCH3, where t is 1-21. For example, t is 14, 15, 16, 17 or 18. In one non-limiting example, t is 16. [0079] In some embodiments of any one of the aspects, R33 is –O(CH2)uR337, where u is 2-10; R337 is C1-C6alkoxy, amino (NH2), CO2H, OH or halo. For example, R337 is -CH3 or NH2. Accordingly, in some embodiments of any one of the aspects described herein, R33 is –O(CH2)u- OMe or R33 is –O(CH2)uNH2. For example, u is 2, 3, 4, 5 or 6. In some embodiments, u is 2, 3 or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6. [0080] In some embodiments of any one of the aspects described herein, R33 is a C1- C6haloalkyl. For example, R33 is a C1-C4haloalkyl. In some embodiments of any one of the aspects described herein, R33 is –CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2. [0081] In some embodiments of any one of the aspects described herein, R33 is – OCH(CH2OR338)CH2OR339 , where R338 and R339 independently are H, optionally substituted C1- C30alkyl, optionally substituted C2-C30alkenyl or optionally substituted C2-C30alkynyl. For example, R338 and R339 independently are optionally substituted C1-C30alkyl. [0082] In some embodiments of any one of the aspects described herein, R33 is – CH2C(O)NHR3310, where R3310 is H, optionally substituted C1-C30alkyl, optionally substituted C2- C30alkenyl or optionally substituted C2-C30alkynyl. For example, R3310 is H or optionally substituted C1-C30alkyl. In some embodiments, R3310 is optionally substituted C1-C6alkyl. [0083] In some embodiments of any one of the aspects described herein, R33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R33 is a reactive phosphorous or a linker covalently attached to a solid support. [0084] In some embodiments of any one of the aspects described herein, R33 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). R35 [0085] In compounds of Formula (III), R35 can be hydroxy, protected hydroxy, phosphate group, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl- ON(CH2R8)(CH2R9), -O-C4-30alkyl-ON(CH2R8)(CH2R9), or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group. [0086] In some embodiments of the various aspects described herein, R35 is R551, optionally substituted C1-6alkyl-R551, optionally substituted -C2-6alkenyl-R551, or optionally substituted -C2- 6alkynyl-R551, where R551 can be –OR552, -SR553, hydrogen, a phosphorous group, a solid support or a linker to a solid support. When R551 is –OR552, R552 can be H or a hydroxyl protecting group. Similarly, when R551 is –SR553, R553 can be H or a sulfur protecting group. [0087] In some embodiments of any one of the aspects described herein, R35 is –OR552 or - SR553. [0088] In some embodiments of any one of the aspects described herein, R552 is a hydroxyl protecting group. Exemplary hydroxyl protecting groups for R552 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). In some embodiments of any one of the aspects described herein, R35 is –OR552 and R552 is 4,4′-dimethoxytrityl (DMT), e.g., R35 is –O-DMT. [0089] In some embodiments of any one of the aspects described herein, R552 is a phosphate group, e.g., R552 is dimethylaminochlorophosphate (-P(O)(NMe2)Cl). [0090] In some embodiments of any one of the aspects described herein, the methylene connecting the R35 to the rest of the compound of Formula (III) is absent and R35 is connected directly to the rest of the compound of Formula (III). [0091] In some embodiments of any one of the aspects described herein, R35 is –CH(R554)- R551, where R554 is hydrogen, halogen, optionally substituted C1-C30alkyl, optionally substituted C2-C30alkenyl, optionally substituted C2-C30alkynyl, or optionally substituted C1-C30alkoxy. [0092] In some embodiments of any one of the aspects, when R35 is –CH(R554)-R551, R554 is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2- C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R554 is H. In some other non-limiting examples, R554 is C1-C30alkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy. [0093] In some embodiments of the various aspects described herein, R35 is –CH(R554)-O-R552, where R554 is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m— (CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R554 is H. In some other non-limiting examples, R554 is C1-C30alkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy. [0094] In some embodiments of the various aspects described herein, R35 is optionally substituted C1-6alkyl-R551 or optionally substituted -C2-6alkenyl-R551, [0095] In some embodiments of any one of the aspects described herein, R35 is – C(R554)=CHR551. It is noted that the double bond in –C(R554)=CHR551 can be in the cis or trans configuration. Accordingly, in some embodiments of any one of the aspects, Rd is – C(R554)=CHR551 and wherein the double bond is in the cis configuration. In some other embodiments of any one of the aspects, Rd is –C(R554)=CHR551 and wherein the double bond is in the trans configuration. [0096] In some embodiments of any one of the aspects described herein, R35 is –CH=CHR551. [0097] In some embodiments of any one of the aspects, when R35 is –C(R554)=CHR551, R554 is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2- C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2—[CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; and R551 is a phosphorous group. For example, R35 is –CH=CHR551. [0098] In some embodiments of any one of the aspects described herein, R551 is a reactive phosphorous group. [0099] In some embodiments of any one of the aspects, R35 is -CH=CH-P(O)(OR555)2, - CH=CH-P(S)(OR555)2, -CH=CH-P(S)(SR556)(OR555), -CH=CH-P(S)(SR556)2, -CH=CH- OP(O)(OR555)2, -CH=CH-OP(S)(OR555)2, -CH=CH-OP(S)(SR556)(OR555), -CH=CH-
OP(S)(SR556)2, -CH=CH-SP(O)(OR555)2, -CH=CH-SP(S)(OR555)2, -CH=CH-
SP(S)(SR556)(OR555), or -CH=CH -SP(S)(SR556)2, where each R555 is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group; and each R556 is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or a sulfur-protecting group.
[00100] In some embodiments of any one of the aspects, at least one R555 in -P(O)(OR555)2, - P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, and -SP(S)(SR556)(OR555) is hydrogen.
[00101] In some other embodiments of any one of the aspects, at least one R555 in -P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, or -SP(S)(SR556)(OR555) is not hydrogen. For example, at least one at least one R555 in P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, - OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, and -SP(S)(SR556)(OR555) is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group.
[00102] In some embodiments of any one of the aspects, at least one R555 is H and at least one R555 is other than H in -P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, - OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, and -SP(S)(SR556)(OR555). [00103] In some embodiments of any one of the aspects, all R555 are H in -P(O)(OR555)2, - P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), - OP(S)(SR556)2, -SP(O)(OR555)2, -SP(S)(OR555)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2.
[00104] In some embodiments of any one of the aspects, all R555 are other than H in in - P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, - OP(S)(SR556)(OR555), -OP(S)(SR556)2, -SP(O)(OR555)2, -SP(S)(OR555)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2.
[00105] In some embodiments of any one of the aspects, at least one R556 in - P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, - SP(S)(SR556)(OR555), and -SP(S)(SR556)2 is H.
[00106] In some embodiments of any one of the aspects, at least one R556 in - P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, - SP(S)(SR556)(OR555), and -SP(S)(SR556)2 is other than H. For example, at least one R556 in - P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, - SP(S)(SR556)(OR555), and -SP(S)(SR556)2 is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an sulfur-protecting group.
[00107] In some embodiments of any one of the aspects, at least one R556 is H and at least one R556 is other than H in -P(S)(SR556)2, -OP(S)(SR556)2 and -SP(S)(SR556)2.
[00108] In some embodiments, all R556 are H in -P(S)(SR556)(OR555), -P(S)(SR556)2, - OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2. [00109] In some embodiments, all R556 are other than H in -P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2. [00110] In some embodiments of any one of the aspects, R35 is -CH=CH-P(O)(OR555)2, where each R555 is H or an oxygen protecting group.
[00111] In some embodiments of any one of the aspects, R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group. For example, R35 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. In other examples, R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group.
[00112] In some embodiments of any one of the aspects described herein, R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
[00113] In some embodiments of any one of the aspects described herein, R35 is a reactive phosphorus group. For example, R33 is -OP(ORP)(N(RP2)2), -OP(SRP)(N(RP2)2), - OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(N(RP2)2), -OP(O)(ORP)H, - OP(S)(ORp)H, -OP(O)(SRp)H, -OP(O)(ORP)RP3, -OP(S)(ORP)RP3, -OP(O)(SRP)RP3, - OP(O)(RP3)(N(RP4)2), or -OP(S)(RP4)(N(RP2)2). [00114] In some embodiments of any one of the aspects, R35 is -OP(ORP)(N(RP2)2), - OP(SRP)(N(RP2)2), -OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(N(RP2)2), - OP(O)(ORP)H, -OP(S)(ORP)H, -OP(O)(RP3)(N(RP4)2), or -OP(S)(RP4)(N(RP2)2), where each RP is independently an optionally substituted C1-6alkyl, each RP2 is independently optionally substituted C1-6alkyl; and each RP3 is independently optionally substituted C1-6alkyl. For example, R35 is OP(O)(RP3)(N(RP4)2), or -OP(S)(RP4)(N(RP2)2). [00115] In some embodiments of any one of the aspects, R35 is –OP(O)(RP4)(N(RP2)2). For example, the R35 is –OP(O)(RP4)(N(RP2)2), where RP4 is Cl and each RP2 is methyl. [00116] In some embodiments of any one of the aspects descried herein, R35is a solid support or a linker covalently attached to a solid support. For example, R35 is –OC(O)CH2CH2C(O)NH-Z, where Z is a solid support. [00117] In some embodiments of any one of the aspects described herein, R33 is H, hydroxyl, protected hydroxyl, nitrogen protecting group, a linker, a ligand or a ligand covalently attached to one or more ligands; and R35 is reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R33 is H or a nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., –OP(O)(RP4)(N(RP2)2). In some embodiment, R33 is a nitrogen protecting group (e.g., trityl); and R35 is –OP(O)(RP4)(N(RP2)2), where RP4 is halogene (e.g., Cl) and each RP2 is independently C1-C6alkyl, e.g., each RP2 is independently methyl. [00118] In some embodiments of any one of the aspects described herein, XM is O, S or CH2; R33 is H, hydroxyl, protected hydroxyl, nitrogen protecting group, a linker, a ligand or a ligand covalently attached to one or more ligands; and R35 is reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support. For example, XM is CH2; R33 is H or a nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., – OP(O)(RP4)(N(RP2)2). In some embodiment, XM is CH2; R33 is a nitrogen protecting group (e.g., trityl); and R35 is –OP(O)(RP4)(N(RP2)2), where RP4 is halogen (e.g., Cl) and each RP2 is independently C1-C6alkyl, e.g., methyl. [00119] In other examples, XM is CH2; R33 is hydroxyl or protected hydroxyl; and R35 is a reactive phosphorous group (e.g., –OP(O)(RP4)(N(RP2)2). In some embodiment, XM is CH2; R33 is protected hydroxyl; and R35 is –OP(O)(RP4)(N(RP2)2), where RP4 is halogen (e.g., Cl) and each RP2 is independently C1-C6alkyl, e.g., methyl. [00120] In some embodiments of any one of the aspects described herein, XM is O; R33 is H or a nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., – OP(O)(RP4)(N(RP2)2). For example, XM is O; R33 is a nitrogen protecting group (e.g., trityl); and R35 is -OP(O)(RP4)(N(RP2)2), where RP4 is halogen (e.g., Cl) and each RP2 is independently Ci- Cealkyl, e.g., methyl.
[00121] In some embodiments of any one of the aspects described herein, XM is O; R33 is hydroxyl or protected hydroxyl and R35 is a reactive phosphorous group (e.g., - OP(O)(RP4)(N(RP2)2). For example, XM is O; R33 is a nitrogen protecting group (e.g., trityl); and R35 is -OP(O)(RP4)(N(RP2)2), where RP4 is halogen (e.g., Cl) and each RP2 is independently Ci- Cealkyl, e.g., methyl.
[00122] In some embodiments of any one of the aspects described herein, XM is S; R33 is H or a nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., - OP(O)(RP4)(N(RP2)2). For example, XM is S; R33 is a nitrogen protecting group (e.g., trityl); and R35 is -OP(O)(RP4)(N(RP2)2), where RP4 is halogen (e.g., Cl) and each RP2 is independently Ci- Cealkyl, e.g., methyl.
[00123] In some embodiments of any one of the aspects described herein, XM is S; R33 is hydroxyl or protected hydroxyl; and R35 is a reactive phosphorous group (e.g., - OP(O)(RP4)(N(RP2)2). For example, XM is S; R33 is a nitrogen protecting group (e.g., trityl); and R35 is -OP(O)(RP4)(N(RP2)2), where RP4 is halogen (e.g., Cl) and each RP2 is independently Ci- Cealkyl, e.g., methyl.
[00124] In some embodiments of any one of the aspects described herein, R33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
[00125] . For example, R33 is a reactive phosphorous or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
[00126] In some embodiments of any one of the aspects described herein, R33 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 R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
[00127] In some embodiments of any one of the aspects described herein, R33 is a solid support, a linker or a linker covalently attached to a solid support; R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
[00128] In some embodiments of any one of the aspects described herein, XM is CEE; R33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. For example, XM is CEE; R33 is a reactive phosphorous or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
[00129] In some embodiments of any one of the aspects described herein, XM is CEE; R33 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 R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e (e.g., E or Z vinylphosphonate) group.
[00130] In some embodiments of any one of the aspects described herein, XM is CEE; R33 is a solid support, a linker or a linker covalently attached to a solid support; R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
[00131] In some embodiments of any one of the aspects described herein, XM is O; R33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. For example, XM is O; R33 is a reactive phosphorous or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
[00132] In some embodiments of any one of the aspects described herein, XM is O; R33 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 R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
[00133] In some embodiments of any one of the aspects described herein, XM is O; R33 is a solid support, a linker or a linker covalently attached to a solid support; R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
[00134] In some embodiments of any one of the aspects described herein, XM is S; R33 is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. For example, XM is S; R33 is a reactive phosphorous or a linker covalently attached to a solid support; and R35 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 R35 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.
[00135] In some embodiments of any one of the aspects described herein, XM is S; R33 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 R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
[00136] In some embodiments of any one of the aspects described herein, XM is S; R33 is a solid support, a linker or a linker covalently attached to a solid support; R35 is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R35 taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.
R43
[00137] In some embodiments of any one of the aspects described herein, R43 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(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or a nitrogen protecting group.
[00138] In some embodiments of any one of the aspects described herein, R43 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.
[00139] In some embodiments of any one of the aspects described herein, R43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
[00140] In some embodiments of any one of the aspects described herein, R43 is a solid support, or a linker (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) covalently bonded to a solid support.
[00141] In some embodiments of any one of the aspects described herein, R43 is hydrogen or a nitrogen protecting group. [00142] In some embodiments of any one of the aspects described herein, R43 is hydroxy or protected hydroxyl. [00143] In some embodiments of any one of the aspects described herein R43 is – P(XD)(N(RP2)2)-R43’, where XD is O or S; each RP2 is independently optionally substituted C1-6alkyl (e.g., methyl); and R43’ is a bond to a subsequent nucleoside, e.g., a bond to 5’ oxygen of a subsequent nucleoside. For example, R43 is –P(O)(N(CH3)2)-R43’. R45 [00144] In some embodiments of any one of the aspects described herein, R45 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 C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R8)(CH2R9), -O-C4-30alkyl- ON(CH2R8)(CH2R9), , monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)- O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P- O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates (R(OH)(O)P-O-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc…), alkyletherphosphonates (R(OH)(O)P-O-5', R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc…), (HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a- P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[-(CH2)a-O- P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[- (CH2)a-O-P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[- (CH2)a-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', wherein a and b are each independently 1-10); or R45 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. [00145] In some embodiments of any one of the aspects described herein, R45 can be a bond to an internucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate (phosphorodithioate), phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, or alkylphosphonates; or R45 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. [00146] In some embodiments of any one of the aspects described herein, R45 is a bond to an internucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C2-30alkenyl, or optionally substituted C1-30 alkoxy; or R45 taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.In some embodiments of any one of the aspects described herein, R45 is a bond to an internucleotide linkage to a preceding nucleotide. [00147] In some embodiments of any one of the aspects described herein, R45 is a hydroxyl or protected hydroxyl. [00148] In some embodiments of any one of the aspects described herein, R45 is optionally substituted C2-30alkenyl or optionally substituted C1-30 alkoxy. [00149] In some embodiments of any one of the aspects described herein, R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group. [00150] In some embodiments of any one of the aspects described herein, the methylene connecting the R45 to the rest of the nucleoside of Formula (VI) is absent and R45 is connected directly to the rest of the nucleoside of Formula (IV). [00151] In some embodiments of any one of the aspects descried herein, R45 is –CH(R451)-X5- R452, where X5 is absent, a bond or O; R451 is hydrogen, optionally substituted C1-30alkyl, optionally substituted -C2-30alkenyl, or optionally substituted -C2-30alkynyl, and R452 is a bond to an internucleoside linkage to the preceding nucleotide. [00152] In some embodiments of any one of the aspects described herein, X5 is O or a bond. For example, X5 is O. In some other embodiments of any one of the aspects described herein, X5 is absent, i.e., R45 is–CH(R451)R452. [00153] In some embodiments of the various aspects described herein, R45 is –CH(R451)-R452 or –C(R451)=CHR452, where R451 is hydrogen, optionally substituted C1-30alkyl, optionally substituted -C2-30alkenyl, or optionally substituted -C2-30alkynyl, and R452 is a bond to an internucleoside linkage to the preceding nucleotide. [00154] In some embodiments of the various aspects described herein, R45 is –CH(R451)-X5- R452. For example, R45 is –CH(R451)-X5-R452 and where R451 is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1- C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(0)- alkyl, C(0)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m— (CH2)P— OH, CH2— [CH(OH)]m— (CH2)P— NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R451 is H. In some other non-limiting examples, R451 is Ci-Csoalkyl optionally substituted with a NH2, OH, C(0)NH2, COOH, halo, SH, or Ci-Cealkoxy.
[00155] In some embodiments of the various aspects described herein, R45 is -CH(R451)-O- R452, where R451 is H or Ci-Csoalkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci-C4)alkyl, N[(Ci-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (Ci-Cs)alkyl, O(Ci-Cs)alkyl (i.e., Ci-Csalkoxy), O(Ci-Cs)haloalkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m— (CH2)P— OH, CH2— [CH(OH)]m— (CH2)P— NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R451 is H. In some other non-limiting examples, R451 is Ci-Csoalkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or Ci-Cealkoxy.
[00156] In some embodiments of any one of the aspects described herein, R45 is - C(R451)=CHR452. It is noted that the double bond in -C(R451)=CHR452 can be in the cis or trans configuration. Accordingly, in some embodiments of any one of the aspects, R45 is - C(R451)=CHR452 and wherein the double bond is in the cis configuration. In some other embodiments of any one of the aspects, R45 is is -C(R451)=CHR452 and wherein the double bond is in the trans configuration. In some embodiments of any one of the aspects described herein, R45 is -CH=CHR452.
[00157] In some embodiments of any one of the aspects described herein, R452 is a bond to an intemucleoside linkage to the preceding nucleotide.
[00158] In embodiments of the various aspects described herein, R45 is optionally substituted Ci-ealkyl-R453, optionally substituted -C2-6alkenyl-R453, or optionally substituted -C2-6alkynyl-R453. In embodiments of the various aspects described herein, R453 can be -OR454, -SR455, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, - OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or - SP(S)(SR457)2; where R454 is hydrogen or oxygen protecting group; R455 is hydrogen or sulfur protecting group; each R456 is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an oxygen-protecting group; and each R457 is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2- soalkenyl, or optionally substituted C2-3oalkynyl, or a sulfur-protecting group.
[00159] In some embodiments of any one of the aspects, at least one R456 in -P(O)(OR456)2, - P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, and -SP(S)(SR457)(OR456) is hydrogen.
[00160] In some other embodiments of any one of the aspects, at least one R456 in -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, or -SP(S)(SR457)(OR456) is not hydrogen. For example, at least one at least one R456 in P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, - OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, and -SP(S)(SR457)(OR456) is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group.
[00161] In some embodiments of any one of the aspects, at least one R456 is H and at least one R456 is other than H in -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, - OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, and -SP(S)(SR457)(OR456). [00162] In some embodiments of any one of the aspects, all R456 are H in -P(O)(OR456)2, - P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), - OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2.
[00163] In some embodiments of any one of the aspects, all R456 are other than H in in - P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, - OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2.
[00164] In some embodiments of any one of the aspects, at least one R457 in - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(S)(SR457)(OR456), and -SP(S)(SR457)2 is H.
[00165] In some embodiments of any one of the aspects, at least one R457 in - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(S)(SR457)(OR456), and -SP(S)(SR457)2 is other than H. For example, at least one R457 in - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(S)(SR457)(OR456), and -SP(S)(SR457)2 is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an sulfur-protecting group.
[00166] In some embodiments of any one of the aspects, at least one R457 is H and at least one R457 IS other than H in -P(S)(SR457)2, -OP(S)(SR457)2 and -SP(S)(SR457)2.
[00167] In some embodiments, all R457 are H in -P(S)(SR457)(OR456), -P(S)(SR457)2, - OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2. [00168] In some embodiments, all R457 are other than H in -P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2. [00169] In some embodiments of any one of the aspects described herein, R45 is optionally substituted -C2-6alkenyl-R453. For example, R45 is -C2-6alkenyl-R453, where C2-6alkenyl is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2— [CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; and R453 is -P(O)(OR456)2, -P(S)(OR456)2, - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), - OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or -SP(S)(SR457)2. [00170] In some embodiments of any one of the aspects, R45 is –CH=CHR453. It is noted that a double bond in the optionally substituted -C2-6alkenyl-R453 can be in the cis or trans configuration. Accordingly, in some embodiments of any one of the aspects, R45 is –CH=CHR453 and wherein the double bond is in the cis configuration. In some other embodiments of any one of the aspects, R45 is –CH=CHR453 and wherein the double bond is in the trans configuration. [00171] In some embodiments of any one of the aspects, R45 is –CH=CH-P(O)(OR456)2, – CH=CH-P(S)(OR456)2, –CH=CH-P(S)(SR457)(OR456), –CH=CH-P(S)(SR457)2, –CH=CH- OP(O)(OR456)2, –CH=CH-OP(S)(OR456)2, –CH=CH-OP(S)(SR457)(OR456), –CH=CH- OP(S)(SR457)2, –CH=CH-SP(O)(OR456)2, –CH=CH-SP(S)(OR456)2, –CH=CH- SP(S)(SR457)(OR456), or –CH=CH -SP(S)(SR457)2. For example, R45 is –CH=CH-P(O)(OR456)2. [00172] In some embodiments, of any one of the aspects, R454 is hydrogen or an oxygen protecting group. For example, R454 is hydrogen or 4,4′-dimethoxytrityl (DMT). In some preferred embodiments, R454 is H. [00173] In some embodiments of any one of the aspects described herein, R45 is optionally substituted –C1-6alkenyl-R453. For example, R45 is –C1-6alkenyl-R453, where C1-6alkenyl is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2— [CH(OH)]m— (CH2)P— OH, CH2— [CH(OH)]m— (CH2)P— NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; and R453 is -OR454, -SR455, -P(O)(OR456)2, - P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, - OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or - SP(S)(SR457)2.
[00174] In some embodiments of any one of the aspects described herein, R45 can be -CH(R458)- R453, where R453 is -OR454, -SR455, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), - P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or -SP(S)(SR457)2; and R458 is H, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl.
[00175] In some embodiments of any one of the aspects described herein, R458 is H or Ci- Csoalkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci-C4)alkyl, N[(Ci-C4)alkyl]2, C(0)NH2, COOH, COOMe, acetyl, (Ci- Cs)alkyl, O(Ci-Cs)alkyl (i.e., Ci-Csalkoxy), O(Ci-Cs)haloalkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)- alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(0H)]m— (CH2)P— OH, CH2— [CH(0H)]m— (CH2)P— NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. In one non-limiting example, R458 is H. In some other non-limiting examples, R458 is Ci-Csoalkyl optionally substituted with a substituent selected from NH2, OH, C(O)NH2, COOH, halo, SH, and Ci-Cealkoxy.
[00176] In some embodiments of any one of the aspects described herein, R45 is -CH(R458)-O- R459, where R459 is H, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, - OP(O)(OR456)2. For example, R45 is -CH(R458)-O-R459, where R458 is H or optionally substituted Ci-C3oalkyl and R459 is H or -P(O)(OR456)2.
[00177] In some embodiments of any one of the aspects described herein, R45 is -CH(R458)-S- R60, where R60 is H, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, - OP(O)(OR456)2.
Ligands
[00166] Without wishing to be bound by a theory, 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.
[00167] 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. Let., 1993, 3, 2765); a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); a phospholipid, e.g., di-hexadecyl-rac-glycerol or tri ethyl ammonium- 1, 2-di-O-hexadecyl-rac- glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); apalmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Then, 1996, 277, 923).
[00168] 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, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, poly aspartate, aptamer, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridines), cross- linkers (e.g. psoralen, mitomycin C), 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, dimethoxytrityl, or phenoxazine), peptides (e.g., an alpha helical peptide, amphipathic peptide, RGD peptide, cell permeation peptide, endosomolytic/fusogenic peptide), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), 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, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, gamma interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high- density lipoprotein (HDL), and a cell-permeation agent (e.g., a.helical cell-permeation agent).
[00169] 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.
[00170] Exemplary 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. [00171] As used herein, the term “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.
[00172] 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 (INF-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).
[00173] Without wishing to be bound by theory, 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-9, 12-dienyl)- 1 ,3 -dioxolan-4- yl)ethanamine (also refered to as XTC herein).
[00174] Synthetic polymers with endosomolytic activity amenable to the present invention are described in U.S. Pat. App. Pub. Nos. 2009/0048410; 2009/0023890; 2008/0287630; 2008/0287628; 2008/0281044; 2008/0281041; 2008/0269450; 2007/0105804; 20070036865; and 2004/0198687, contents of which are hereby incorporated by reference in their entirety.
[00175] 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 (indohcidin, SEQ ID NO: 226); AAVALLPAVLLALLAP (RFGF, SEQ ID NO: 227); AALLPVLLAAP (RFGF analogue, SEQ ID NO: 228); and RKCRIVVIRVCR (bactenecin, SEQ ID NO: 229).
[00176] Exemplary cationic groups include, but are not limited to, protonated amino groups, derived from e.g., O- AMINE (AMINE = NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH2)nAMINE, (e.g., AMINE = NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); and NEI(CEECEENEI)nCEECEE-AMINE (AMINE = NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).
[00177] As used herein the term “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.
[00178] 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.
[00179] A number of folate and folate analogs amenable to the present invention as ligands are described in U.S. Pat. Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, contents of which are herein incorporated in their entireties by reference. [00180] As used herein, the terms “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). 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). 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. In addition, aptamers that bind serum components (e.g. serum proteins) are also amenable to the present invention as PK modulating ligands. Binding to serum components (e.g. serum proteins) can be predicted from albumin binding assays, scuh as those described in Oravcova, et al., Journal of Chromatography B (1996), 677: 1-27.
[00181] When two or more ligands are present, the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties. For example, a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In a preferred embodiment, all the ligands have different properties.
[00182] In some embodiments of any one of the aspects, the ligand has a structure shown in any of Formula (IV) - (VII):
Figure imgf000042_0001
wherein: q2A, q2B, q3 \ q3l\ q4 \ q4B, q5A, q5B and q5C represent independently for each occurrence
0-20 and wherein the 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;
Q2A Q2B Q3A Q3B Q4A Q4B Q5A QSB Q5C are mdependently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R’)=C(R”), C=C or C(O);
R2A R2B, R3A R3B R4A R4B, R5A RSB R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=N-O,
Figure imgf000042_0002
heterocyclyl;
L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, tri saccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and
Ra is H or amino acid side chain.
[00183] In some embodiments of any one of the aspects, the ligand is of Formula (VII):
Figure imgf000043_0001
wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.
[00184] Exemplary ligands include, but are not limited to, the following:
Figure imgf000043_0002
Figure imgf000044_0001
Ligand 7
Figure imgf000045_0001
Ligand 8.
[00185] In some embodiments of any one of the aspects described herein, 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.
[00186] In some embodiments, the ligand can be a tri-antennary ligand described in Figure 3 of US Patent No. 6,906,182. For example, the ligand is selected from the following tri-antennary ligands:
Figure imgf000046_0001
[00187] It is noted that when more than one ligand are present, they can be 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.
[00188] In some embodiments of any one of the aspects described herein, the ligand is selected from the group consistof ligands shown in FIG. 27.
Linkers
[00189] Embodiments of the various aspects described herein include a linker. As used herein, the term “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 NR1, C(O), C(O)O, C(O)NR1, 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, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl. alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R1)2, C(O), cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R1 is hydrogen, acyl, aliphatic or substituted aliphatic.
[00190] In some embodiments, 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. As such, 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). [00191] Generally, 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. In a preferred embodiment, 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).
[00192] 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. Examples of such 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.
[00193] 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.
[00194] 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.
[00195] In general, 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. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, 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).
[00196] One class of cleavable linking groups is 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-). To determine if 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. For example, 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. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, 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.
[00197] 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. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of 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(S)(Rk)- O-, -S-P(O)(Rk)-S-, -O-P(S)( Rk)-S-, wherein Rk at each occurrence can be, independently, hydrogen, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, C7-C12 aralkyl. 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(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using methods analogous to those described above.
[00198] 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. In preferred embodiments 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. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). 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. These candidates can be evaluated using methods analogous to those described above.
[00199] 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. Examples of 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.
[00200] 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 - NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of the two adjacent amino acids.
[00201] In some embodiments of any one of the aspects described herein, the linker is - C(O)CH2CH2C(O)-, -OC(O)CH2CH2C(O)-, -OC(O)CH2CH2C(O)O-, -C(O)CH2CH2C(O)NH- or -OC(O)CH2CH2C(O)NH-. For example, the linker is -OC(O)CH2CH2C(O)NH-
Internucleoside linkages [00202] As used herein, “intemucleoside linkage” 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. Representative phosphorus containing linkages include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative 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. In certain embodiments, 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.
[00203] 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. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, 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. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral. In other words 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).
[00204] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers. Thus, while not wishing to be bound by theory, 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).
[00205] 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). The replacement can occur at the either one of the linking oxygens or at both linking oxygens. When the bridging oxygen is the 3 ’-oxygen of a nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5 ’-oxygen of a nucleoside, replacement with nitrogen is preferred.
[00206] 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.”
[00207] In certain embodiments, 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.
[00208] Examples of moieties which can replace the phosphate group include, but are not limited to, amides (for example amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)- C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methyl enemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’, C3’- CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5’ and nonionic linkages containing mixed N, O, S and CH2 component parts. See for example, Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amides, carbamate and ethylene oxide linker.
[00209] One skilled in the art is well aware that in certain instances replacement of a non- bridging oxygen can lead to enhanced cleavage of the intersugar linkage by the neighboring 2’- OH, thus in many instances, 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.
[00210] 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.
[00211] Additional exemplary non-phosphorus containing intemucleoside linking groups are described in U.S. Patent Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;
5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;
5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, content of each of which is incorporated herein by reference.
[00212] In some embodiments of any one of the aspects, the oligonucleotides described herein comprise one or more neutral intemucleoside linkages that are non-ionic. Suitable neutral intemucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2- C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3 '-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'); nonionic linkages containing siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and/or amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)); and nonionic linkages containing mixed N, O, S and CEE component parts.
[00213] In one embodiment, the non-phosphodiester backbone linkage is selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate and phosphoramidate backbone linkages.
[00214] In some embodiments of any one of the aspects described herein, the intemucleoside linkage
Figure imgf000053_0001
where RIL1 and RIL2 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 RIL3 and RIL4 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. an alkyl group, an aryl group, etc... ), H, NR2 (R is hydrogen, alkyl, aryl), alkyl or aryl. It is understood that one of RIL1 and RIL2 is replacing the oxygen linked to 5’ carbon of a first nucleoside sugar and the other of RIL1 and RIL2 is replacing the oxygen linked to 3’ (or 2’) carbon of a second nucleoside sugar.
[00215] In some embodiments of any one of the aspects, RIL1, RIL2, RIL3 and RIL4 all are O.
[00216] In some embodiments, RIL1 and RIL2 are O and at least one of RIL3 and RIL4 is other than
O. For example, one of RIL3 and RIL4 is S and the other is O or both of RIL3 and RIL4 are S.
[00217] In some embodiments of any one of the aspects described herein, one of R43 or R45 is a bond to a modified intemucleoside linkage, e.g., an intemucleoside linkage of structure:
Figure imgf000054_0001
where at least one of RIL1, RIL2, RIL3 and RIL4 is not O. For example, at least one of RIL3 and RIL4 is S.
[00218] In some embodiments of any one of the aspects described herein, both of R43 and R45 are a bond to a modified intemucleoside linkage.
[00219] In some embodiments of any one of the aspects described herein R43 is a bond to phosphodiester intemucleoside linkage.
[00220] In some embodiments of any one of the aspects described herein R45 is a bond to phosphodiester intemucleoside linkage.
[00221] In some embodiments of any one of the aspects described herein, R43 is a bond to a modified intemucleoside linkage and R45 is a bond to phosphodiester intemucleoside linkage.
[00222] In some embodiments of any one of the aspects described herein, R45 is a bond to a modified intemucleoside linkage and R43 is a bond to phosphodiester intemucleoside linkage.
[00223] In some embodiments of any one of the aspects described herein, the intemucleotide linkage is -P(XD)(N(Rp)2)-, where XD is O or S; and each RP2 is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl.
[00224] In some embodiments of any one of the aspects described herein, R43 is linked to an intemucleotide linkage of formula -P(XD)(N(Rp)2)-@, where XD is O or S; each RP2 is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl; and @ is a bond to 5 ’-position of a subsequent nucleoside. For example, R43 is linked to an intemucleotide linkage of formula -P(XD)(N(Rp)2)-@, where XD is O or S; each RP2 is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl; and @ is a bond to R45 of a subsequent nucleoside of Formula (IV). [00225] In some embodiments of any one of the aspects, the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more modified intemucleoside linkages. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5 or 6 modified intemucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3 or 4 modified intemucleoside linkages. In some embodiments, 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. For example, 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.
[00226] In some embodiments of any one of the aspects, the modified intemucleoside linkage is a phosphorothioate. Accordingly, in some embodiments of any one of the aspects, the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate intemucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3, 4, 5 or 6 phosphorothioate intemucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3 or 4 phosphorothioate intemucleoside linkages. In some embodiments, 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. For example, 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
[00227] Some embodiments of the various aspects described herein include an oxygen protecting group (also referred to as an hydroxyl protecting group herein). Oxygen protecting groups include, but are not limited to, -ROP1, -N(ROP2)2, -C(=O)SRopl, -C(=O)Ropl, -CO2Ropl, -C(=O)N(ROP2)2, -C(=NROP2)ROP1, -C(=NR°P2)OROP1, -C(=NROP2)N(ROP2)2, -S(=O)ROP1, -SO+ 2ROP1, -SI(ROP1)3, -P(ROP3)2, -P(ROP3)+3 X , -P(OROP3)2, -P(OROP3)3 X , -P(=O)(ROP1)2, -P(=O)(OR°P3)2, and -P(=O)(N(ROP2)2)2; wherein each X is a counterion; each ROP1 is independently Ci-io alkyl, Ci-io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi-io alkyl, heteroC2-ioalkenyl, heteroC2-ioalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, or 5-14 membered heteroaryl, or two ROP1 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; each ROP2 is hydrogen, -OH, -ORopl, -N(ROP3)2, -CN, -C(=O)Ropl, -C(=O)N(ROP3)2, -CO2ROP1, -SO2ROP1, -C(=NR°P3)OROP1, -C(=NROP3)N(ROP3)2, -SO2N(ROP3)2, -SO2ROP3, -SO2OR°P3, -SOROP1, -C(=S)N(ROP3)2, -C(=O)SROP3, -C(=S)SROP3, -P(=O)(ROP1)2, -P(=O)(OROP3)2, -P(=O)(N(ROP3)2)2, CI-10 alkyl, Ci-io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi- walkyl, heteroC2-ioalkenyl, heteroC2-ioalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two ROP2 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each ROP3 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi- 10 alkyl, heteroC2-io alkenyl, heteroC2-io alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two ROP3 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of ROP1, ROP2 and ROP3 can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci-C4)alkyl, N[(Ci-C4)alkyl]2, C(0)NH2, COOH, COOMe, acetyl, (Ci-Cs)alkyl, O(Ci- Csjalkyl (i.e., Ci-Csalkoxy), O(Ci-Cs)haloalkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(0)- alkyl, C(0)- alkyl, alkylcarbonylaminyl, CH2 — [CH(0H)]m— (CH2)P— OH, CH2— [CH(0H)]m— (CH2)P— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00228] 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, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
[00229] Exemplary 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- methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, 1 -[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4- yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro- 7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1 -ethoxy ethyl, 1 -(2-chloroethoxy)ethyl, 1-methyl-l- methoxy ethyl, 1 -methyl- 1 -benzyloxy ethyl, 1- methyl- 1 -benzyloxy-2-fluoroethyl, 2,2,2- trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o- nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2- picolyl, 4-picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4, 4', 4"- tris(benzoyloxyphenyl)methyl, 3-(imidazol-l- yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-l'-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, l,3-benzodisulfuran-2-yl, benzisothiazolyl S,S- dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p- chlorophenoxyacetate, 3 -phenylpropionate, 4- oxopentanoate (levulinate), 4,4-
(ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9- fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2- (triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4- (1,1 ,3,3-tetramethylbutyl)phenoxyacetate, 2,4- bis( 1 , 1 -dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuSP3inoate, (E)-2-methyl-2-butenoate, o- (methoxyacyl)benzoate, a-naphthoate, nitrate, alkylN,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).
[00230] In some embodiments of any one of the aspects described herein, 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). In certain embodiments, 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. [00231] The terms “protected hydroxyl” and “protected hydroxyl” as used herein mean a group of the formula -ORPro, wherein RPro 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 include, but are not limited to, -OH, -ORNP1, -N(RNP2)2, -C(=O)RNP1, -C(=O)N(RNP2)2, -CO2RNP1, - SO2RNP1, -C(=NRNP2)RNP1, -C(=NRNP2)ORNP1, -C(=NRNP2)N(RNP2)2, -SO2N(RNP2)2, -SO2RNP2, - SO2ORNP2, -SORNP1, -C(=S)N(RNP2)2, -C(=O)SRNP2, -C(=S)SRNP2, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6- 14 aryl, and 5-14 membered heteroaryl groups, where each RNP1 is independently C1-10 alkyl, C1- 10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10alkenyl, heteroC2- 10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, or 5-14 membered heteroaryl, or two RNP1 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RNP2 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2- 10 alkynyl, heteroC1-10 alkyl, heteroC2-10 alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP3 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of RNP1 and RNP2 can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2—C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2—C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2— [CH(OH)]m—(CH2)p—OH, CH2—[CH(OH)]m—(CH2)p—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. [00233] 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, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference. [00234] Exemplary amide (e.g., -C(=O)RNP1) nitrogen protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3 -pyridylcarboxamide, N- benzoylphenylalanyl derivative, benzamide, p- phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'- dithiobenzyloxy acylamino)acetamide, 3-(p- hy droxylphenyl)propanamide, 3 -(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o- phenylazophenoxy)propanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o- nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.
[00235] Exemplary carbamate (e.g., -C(=O)ORNP1) nitrogen protecting groups include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (l-adamantyl)-l-methylethyl carbamate (Adpoc), l,l-dimethyl-2-haloethyl carbamate, 1 , 1 -dimethyl-2,2-dibromoethyl carbamate (DB-t- BOC), l,l-dimethyl-2, 2, 2-tri chloroethyl carbamate (TCBOC), 1 -methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-t- butylphenyl)-! -methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'- pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1- isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxylpiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p- bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2- methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p- toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), 4- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2- phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), 1,1- dimethyl-2-cyanoethyl carbamate, m- chloro-p-acyloxybenzyl carbamate, p-(dihydroxylboryl)benzyl carbamate, 5- benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)- 6-chromonylmethyl carbamate (Tcroc), m- nitrophenyl carbamate, 3, 5 -dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy- 6-nitrobenzyl carbamate, phenyl(o- nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2- dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3- (N,N- dimethylcarboxamido)propyl carbamate, 1 , 1 -dimethylpropynyl carbamate, di (2- pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1 -methylcyclobutyl carbamate, 1 -methylcyclohexyl carbamate, 1 -methyl- 1- cyclopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxyphenyl)ethyl carbamate, 1- methyl-l-(p- phenylazophenyl)ethyl carbamate, 1-methyl-l -phenylethyl carbamate, 1- methyl-l-(4- pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6- trimethylbenzyl carbamate.
[00236] Exemplary sulfonamide (e.g., -S(=O)2RNP1) nitrogen protecting groups include, but are not limited to, such as p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, - trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4- methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4- methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4- methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), 0- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
[00237] 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-one, 1- substituted 3,5-dinitro-4-pyridone, N-methylamine, N- allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3 -acetoxypropylamine, N-(l- isopropyl-4- nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N- di(4- methoxyphenyl)methyl amine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N- [(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fem), N-2- picolylamino N'- oxide, N- 1,1 -dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl] methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'- isopropylidenediamine, N-p- nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2- hydroxylphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l- cyclohexenyl)amine, N-borane and N-diphenylborinic acid derivative, N- [phenyl(pentNP1cylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N- nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o- nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4- methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3- nitropyridinesulfenamide (Npys). Sulfur protecting groups [00238] Some embodiments of the various aspects described herein include sulfur protecting group (also referred to as a thiol protecting group herein). Sulfur protecting groups include, but are not limited to, -RSP1, -N(RSP2)2, -C(=O)SRSP1, -C(=O)RSP1, -CO2RSP1, −C(=O)N(RSP2)2, - C(=NRSP2)RSP1, -C(=NRSP2)ORSP1, -C(=NRSP2)N(RSP2)2, -S(=O)RSP1, -SO2RSP1, −Si(RSP1)3, - P(RSP3)2, -P(RSP3)+3 X−, -P(ORSP3)2, -P(ORSP3)+3 X−, -P(=O)(RSP1)2, -P(=O)(ORSP3)2, and−P(=O)(N(RSP2) 2)2, wherein [00239] X- is a counterion; each RSP1 is independently C1-10 alkyl, C1-10 perhaloalkyl, C2- 10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3- 10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, or 5-14 membered heteroaryl, or two RSP1 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; each RSP2 is hydrogen, −OH, −ORSP1, −N(RSP3)2, −CN, −C(=O)RSP1, −C(=O)N(RSP3)2, −CO2RSP1, −SO2RSP1, −C(=NRSP3)ORSP1, −C(=NRSP3)N(RSP3)2, −SO2N(RSP3)2, −SO2RSP3, −SO2ORSP3, −SORSP1, −C(=S)N(RSP3)2, −C(=O)SRSP3, −C(=S)SRSP3, −P(=O)(RSP1)2, −P(=O)(ORSP3)2, −P(=O)(N(RSP3)2)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP2 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RSP3 is independently hydrogen, C1- 10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10 alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP3 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of RSP1, RSP2 and RSP3 can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8alkoxy), O(C1- C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m — (CH2)P — OH, CH2 — [CH(OH)]m — (CH2)P — NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00240] 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, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
[00241] It is noted that the 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, R43 is a hydroxyl or protected hydroxyl group. In some embodiments, when the nucleoside of Formula (IV) is present at the 3’- terminus of the oligonucleotide, R43 is a hydroxyl. In other embodiments, when the nucleoside of Formula (IV) is present at the 3 ’-terminus of the oligonucleotide, R43 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, R43 is a hydrogen.
[00242] In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (IV), a nucleoside with a modified sugar. By a “modified sugar” is meant a sugar or moiety other than 2’-deoxy (i.e, 2’-H) or 2’-OH ribose sugar. Some exemplary 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), threose (Threose nucleic acid, TNA), and 2,3 -dihydroxylpropyl (glycol nucleic acid, GNA). It is noted that the nucleoside with the modified sugar can be present at any position of the oligonucleotide.
[00243] In some embodiments, 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. For example, 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.
[00244] In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), and 2’-F nucleosides.
[00245] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-0Me nucleotides. For example, 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.
[00246] In some embodiments, 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.
[00247] In some embodiments, 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. For example, 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. For example, 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. In some embodiments, the oligonucleotide comprises a 2’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the oligonucleotide.
[00248] In some embodiments, 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. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), 2’-OMe nucleosides, 2’-F nucleosides and 2’-deoxy (2’-H) nucleotides.
[00249] In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (IV), a non-natural nucleobase. In some embodiments, 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. When present, a nucleotide comprising a non-natural nucleobase can be present anywhere in the oligonucleotide. [00250] In some embodiments, the oligonucleotide further comprises a solid support linked thereto.
[00251] 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. For example, the oligonucleotide can be from 5 nucleotides to 100 nucleotides in length. In some embodiments, the oligonucleotide is from 10 nucleotides to 50 nucleotides in length. For example, 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. 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.
[00252] In some embodiments, the oligonucleotide described herein comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 intemucleotidic linkages in the Sp configuration. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
[00253] In some embodiments, the oligonucleotide described herein comprises a stereochemistry block. In some embodiments, a block is an Rp block in that each intemucleotidic linkage of the block is Rp. In some embodiments, a 5 ’-block is an Rp block. In some embodiments, a 3 ’-block is an Rp block. In some embodiments, a block is an Sp block in that each intemucleotidic linkage of the block is Sp. In some embodiments, a 5 ’-block is an Sp block. In some embodiments, a 3 ’-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, 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.
[00254] In some embodiments, the oligonculeotide described herein comprises a 5’-block is an Sp block wherein each sugar moiety comprises a 2 ’-fluoro modification. In some embodiments, 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. In some embodiments, 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. In some embodiments, a 5’-block comprises 4 or more nucleoside units. In some embodiments, 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. In some embodiments, 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. In some embodiments, a 3 ’-block comprises 4 or more nucleoside units. In some embodiments, a 3 ’-block comprises 5 or more nucleoside units. In some embodiments, a 3 ’-block comprises 6 or more nucleoside units. In some embodiments, a 3 ’-block comprises 7 or more nucleoside units.
[00255] In some embodiments, 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. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.
[00256] In some embodiments of any one of the aspects described herein, 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-51- (HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P- 0-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 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. vinyl, substituted vinyl, e.g., OH)2(O)P-5'-CH= or (OH)2(O)P-5'-CH2-), 5'- alkyletherphosphonates (e.g., R(0H)(0)P-0-5', R=alkylether, e.g., methoxymethyl (MeOCH2-), ethoxymethyl, etc.) Other 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[-(CH2)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[-(CH2)a-O-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[- (CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[-(CH2)a-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', wherein a and b are each independently 1-10. Other embodiments, include replacement of oxygen and/or sulfur with BH3, BH?" and/or Se.
[00257] In some embodiments of any one of the aspects described herein, the oligonucleotide comprises a 5’-vinylphosphonate group (i.e., the 4’-C of the 5’-terminal nucleotide is bonded to a vinyl phosphonate). For example, the oligonucleotide comprises a 5’-E-vinyl phosphonate group. In some other non-limiting example, the oligonucleotide comprises a 5’-Z-vinylphosphonate group. [00258] In some embodiments of any one of the aspects, 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. [00259] In some embodiments of any one of the aspects, 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. For example, 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). In some embodiments, 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. In some embodiments, thermally destabilizing modification is located in positions 2-9, or preferably positions 4-8, counting from the 5 ’-end of the oligonucleotide. In some further embodiments, 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.
[00260] The term “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). In some embodiments, 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.
[00261] 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). For example, the thermally destabilizing modifications can include, but are not limited to, mUNA and GNA building blocks as follows:
Figure imgf000069_0001
Figure imgf000070_0001
[00262] In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA.
[00263] In some embodiments, the destabilizing modification mUNA is selected from the group consisting of
Figure imgf000071_0001
- 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.
[00264] In some embodiments, the destabilizing modification mUNA is selected from the group consisting of
Figure imgf000071_0002
; 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.
[00265] In some embodiments, the destabilizing modification mUNA is selected from the group consisting of
Figure imgf000072_0001
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.
[00266] In some embodiments, the destabilizing modification mUNA is selected from the group consisting of
Figure imgf000073_0001
R = H, OH; OMe; Cl, F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; 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
[00267] In some embodiments, the destabilizing modification mUNA is selected from the group consisting of
Figure imgf000073_0002
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
[00268] In some embodiments, the modification mUNA is selected from the group consisting of
Figure imgf000074_0001
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 [00269] Exemplary abasic modifications include, but are not limited to the following:
Figure imgf000075_0002
Mod3 Mod4 Mod5 (2'-OMe Abasic (3'-0Me) (5'-Me) (Hyp-spacer) Spacer)
X = OMe, F wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.
[00270] Exemplified sugar modifications include, but are not limited to the following:
Figure imgf000075_0001
R= H, OH, O-alkyl R'" = H, OH, CH3, CH2CH3, O-alkyl, NH2, NHMe, NMe2 R"" = H, OH, CH3, CH2CH3, O-alkyl, NH2, NHMe, NMe2 wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic. [00271] In some embodiments the thermally destabilizing modification of the duplex is selected from the mUNA and GNA building blocks described in Examples 1-3 herein. In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA. In some further embodiments of this, 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).
[00272] The term “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. In some
Figure imgf000076_0001
, 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. In one example, 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). In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). 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.
[00273] The term ‘GNA’ 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:
Figure imgf000077_0001
(R)-GNA
[00274] 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. In certain embodiments, 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.
[00275] In some embodiments, 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:
Figure imgf000078_0001
[00276] More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.
[00277] 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.
[00278] In some embodiments, 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. These 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:
Figure imgf000078_0002
inosine nebularine 2-aminopurine
2
Figure imgf000078_0003
,4- difluorotoluene 5-nitroindole 3-nitropyrrole 4-Fluoro-6- 4-Methylbenzimidazole methylbenzimidazole [00279] In some embodiments, 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:
Figure imgf000079_0001
wherein R is H, OH, OCH3, F, NH2, NHMe, NM02 or O-alkyl
[00280] Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:
Figure imgf000079_0002
[00281] 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.
[00282] In some embodiments of any one of the aspects described herein, the oligonucleotide can comprise one or more stabilizing modifications. For example, the oligonucleotide can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
[00283] In some embodiments, the oligonucleotide comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the oligonucleotide can be present at any positions. In some embodiments, the oligonucleotide comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16, counting from the 5 ’-end. In some other embodiments, the oligonucleotide comprises stabilizing modifications at positions 2, 6, 14 and 16, counting from the 5 ’-end. In still some other embodiments, the oligonucleotide comprises stabilizing modifications at positions 2, 14 and 16, counting from the 5 ’-end. In some embodiments, 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.
[00284] In some embodiments, the oligonucleotide comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, 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. In some embodiments, 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.
[00285] In some embodiments, 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.
[00286] Exemplary thermally stabilizing modifications include, but are not limited to 2’-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to LNA.
Double-stranded RNAs
[00287] The skilled person is well aware that double-stranded 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. [00288] Accordingly, in one aspect, provided herein is a double-stranded RNA (dsRNA) 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. In other words, 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),
[00289] In some embodiments of any one of the aspects described herein, the sense strand is an oligonucleotide described herein. In other words, the sense strand comprises at least one nucleotide of Formula (IV),
[00290] In some embodiments of any one of the aspects described herein, the antisense strand is an oligonucleotide described herein. In other words, the antisense strand comprises at least one nucleotide of Formula (IV).
[00291] In some embodiments of the various aspects described herein, 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.
[00292] Each strand of the dsRNA molecule can range from 15-35 nucleotides in length. For example, 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. Without limitations, the sense and antisense strands can be equal length or unequal length. For example, the sense strand and the antisense strand independently have a length of 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
[00293] In some embodiments, 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. For example, the antisense strand can be 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. In some embodiments, the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length. In some particular embodiments, the antisense strand is 22, 23 or 24 nucleotides in length. For example, the antisense strand is 23 nucleotides in length.
[00294] Similar to the antisense strand, 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. For example, 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
35 nucleotides in length. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides in length. In some particular embodiments, the sense strand is 20, 21 or 22 nucleotides in length. For example, the sense strand is 21nucleotides in length.
[00295] In some embodiments, 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. In some embodiments, 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, and 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. For example, 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. In some embodiments, the sense strand and the antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, 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. In some particular embodiments, the sense strand is 20, 21 or 22 nucleotides in length and the antisense strand is 22, 23 or 24 nucleotides in length. For example, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.
[00296] The sense strand and antisense strand typically form a double-stranded or duplex region. Without limitations, the duplex region of a dsRNA agent described herein can be 12-35 nucleotide (or base) pairs in length. For example, 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. In another example, 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.
[00297] As described herein, 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. [00298] In some embodiments, 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. For example, the nucleotide of Formula (IV) described herein can be present at a terminal region of the sense strand. For example, the nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3 and
4, counting from the 5 ’-end of the sense strand. In another non-limiting example, the 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.
[00299] In some embodiments, 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. For example, the nucleotide of Formula (IV) described herein can be present at a terminal region of the antisense strand. For example, the nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3, 4,
5, 6, 7, 8, and 9, counting from the 5’-end of the antisense strand. In another non-limiting example, the 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.
[00300] In some embodiments, 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. For example, 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. In some embodiments, 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.
[00301] As described herein, 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. A nucleotide comprising modified sugar can be present anywhere in the dsRNA molecule. For example, 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. When 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.
[00302] As described herein, 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. In some embodiments, 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. For example, the sense strand comprises a 2 ’-fluoro nucleotide at position 10, counting from 5 ’-end of the sense strand. In some embodiments, 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. For example, the sense strand comprises a 2 ’-fluoro nucleotide at positions 9 10, counting from 5 ’-end of the sense strand. In another example, the sense strand comprises a 2’-fluoro nucleotide at positions 10 and 11, counting from 5 ’-end of the sense strand. In some embodiments, 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.
[00303] In some embodiments, the antisense comprises 2 ’-fluoro nucleotides at positions 7, 10 and 11 from the 5’-end. In some other embodiments, 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). In some embodiments, 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. In some other embodiments, 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. For example, 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.
[00304] In some other embodiments, 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 . For example, 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.
[00305] In some embodiments, the sense strand comprises a block of two, three or four 2’- fluoro nucleotides. For example, 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).
[00306] In some embodiments, 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.
[00307] In some embodiments, 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. For example, the antisense strand can comprise a 2’-fluoro nucleotide at position 14, counting from 5 ’-end of the antisense strand. In some embodiments, the antisense comprises 2 ’-fluoro nucleotides at positions 2 and 14, counting from the 5 ’-end of the antisense strand. In some embodiments, the antisense comprises 2’-fluoro nucleotides at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, 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.
[00308] In some embodiments, the antisense strand comprises at least one 2’-fluoro nucleotide adjacent to a destabilizing modification. For example, 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. In some embodiments, 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. In some embodiments, 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.
[00309] In some embodiments, 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. For instance, 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.
[00310] As described herein, 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. Without limitations, 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.
[00311] In some embodiments, 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. In some embodiments, 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. [00312] As described herein, 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. For example, 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.
[00313] As described herein, 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. For example, 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.
[00314] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5 or 6 of 2’-deoxy nucleotides. For example, 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. For example, 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. In one non-limiting example, 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.
[00315] In some embodiments, the antisense comprises a 2 ’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the antisense strand. For example, the antisense strand comprises a 2’- deoxy nucleotide at positions 5, 7 and 12, counting from 5 ’-end of the antisense strand. In some embodiments, the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5 and 7, counting from 5’-end of the antisense strand. For example, the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7 and 12, counting from 5’-end of the antisense strand. In some embodiments, the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7, 12 and 14, counting, from 5’-end of the antisense strand. For example, 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] In some embodiments, the antisense comprises a 2’-deoxy nucleotide at position 2 or 12, counting from 5’-end of the antisense strand. For example, the antisense comprises a 2’-deoxy nucleotide at position 12, counting from 5 ’-end of the antisense strand. [00317] In some embodiments, 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.
[00318] In some embodiments, 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.
[00319] In some embodiments, 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.
[00320] In some embodiments, 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.
[00321] In one non-limiting example, the sense strand does not comprise a 2 ’-deoxy nucleotide at position 11, counting from 5 ’-end of the sense strand.
[00322] In some embodiments, 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
[00323] A nucleotide comprising a non-natural nucleobase can be present anywhere in the dsRNA molecule. For example, 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. When 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.
[00324] 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. For instance, 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.
[00325] In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate intemucleoside linkage modification in the overhang region. For example, 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. For example, at least 2, 3, 4, or all 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. For instance, there may be at least two phosphorothioate intemucleoside linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3 ’-end of the antisense strand.
[00326] In some embodiments, 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.
[00327] In some embodiments, 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.
[00328] In some embodiments, 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.
[00329] In some embodiments, 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.
[00330] In some embodiments, 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.
[00331] In some embodiments, 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.
[00332] In some embodiments, 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. [00333] In some embodiments, 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.
[00334] In some embodiments, 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.
[00335] In some embodiments, 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. For example, 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.
[00336] In some embodiments, 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. For example, 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).
[00337] In some embodiments, 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).
[00338] In some embodiments, 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).
[00339] In some embodiments, 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).
[00340] In some embodiments, 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).
[00341] In some embodiments, 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).
[00342] In some embodiments, 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).
[00343] In some embodiments, 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). [00344] In some embodiments, 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).
[00345] In some embodiments, 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).
[00346] In some embodiments, 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).
[00347] In some embodiments, 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).
[00348] In some embodiments, 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).
[00349] In some embodiments, 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).
[00350] In some embodiments, 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).
[00351] In some embodiments, 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).
[00352] In some embodiments, 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).
[00353] In some embodiments, 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).
[00354] In some embodiments, 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).
[00355] In some embodiments, the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand. For example, 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.
[00356] In some embodiments, the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’ -end of the antisense strand. For example, 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. [00357] In some embodiments, the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’ end of the antisense strand. For example, 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. In other words, 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.
[00358] In some embodiments, 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. For example, 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.
[00359] In some embodiments, 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. For example, 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, and 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.
[00360] In some embodiments, 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. For example, 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, and 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.
[00361] In some embodiments, dsRNA molecule described herein comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 intemucleotidic linkages in the Sp configuration. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.
[00362] In some embodiments, dsRNA molecule described herein comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each intemucleotidic linkage of the block is Rp. In some embodiments, a 5 ’-block is an Rp block. In some embodiments, a 3 ’-block is an Rp block. In some embodiments, a block is an Sp block in that each intemucleotidic linkage of the block is Sp. In some embodiments, a 5 ’-block is an Sp block. In some embodiments, a 3 ’-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, 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.
[00363] In some embodiments, dsRNA molecule described herein comprises a 5 ’-block is an Sp block wherein each sugar moiety comprises a 2 ’-fluoro modification. In some embodiments, 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. In some embodiments, 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. In some embodiments, a 5’-block comprises 4 or more nucleoside units. In some embodiments, 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. In some embodiments, 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. In some embodiments, a 3 ’-block comprises 4 or more nucleoside units. In some embodiments, a 3 ’-block comprises 5 or more nucleoside units. In some embodiments, a 3 ’-block comprises 6 or more nucleoside units. In some embodiments, a 3 ’-block comprises 7 or more nucleoside units.
[00364] In some embodiments, 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. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.
[00365] Various publications describe multimeric siRNA which can all be used with the oligonucleotide and dsRNA of the invention. Such publications include W02007/091269, US Patent No. 7858769, W02010/141511, W02007/117686, W02009/014887 and WO2011/031520 which are hereby incorporated by their entirely.
[00366] In some embodiments, 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. For example, the overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In some embodiments, 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.
[00367] In some embodiments, 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. For example, 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.
[00368] The 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule described herein may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, 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.
[00369] 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. For example, 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.
[00370] Generally, 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. For example, the single overhang is at least one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in length. In some embodiments, 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. [00371] 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.
[00372] As 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. By way of example, 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. For example, 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.
[00373] It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5’ or 3’ overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3’ or 5’ overhang may be modified, e.g., with a modification described herein. 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. [00374] In some embodiments, the dsRNA molecule described herein comprises modifications of an alternating pattern, particular in the Bl, B2, B3, Bl’, B2’, B3’, B4’ regions. The term “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. For example, if 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 ... ,”
“AAABBBAAABBB. .. ,” or “AB CAB CAB CAB C... ,” etc.
[00375] The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, 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.
[00376] In some embodiments, 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. For example, 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. As another example, 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.
[00377] In some embodiments of any one of the aspects described herein, 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 '-monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 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. vinyl, substituted vinyl, e.g., OH)2(O)P-5'- CH= or (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (e.g., R(0H)(0)P-0-5', R=alkylether, e.g., methoxymethyl (MeOCH2-), ethoxymethyl, etc.) Other 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[-(CH2)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: H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O- P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', HO[- (CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[-(CH2)a-P(X)(OH)-O]b- 5', Me2N[- (CH2)a-P(X)(OH)-O]b- 5', wherein a and b are each independently 1-10. Other embodiments, include replacement of oxygen and/or sulfur with BH3, BHV and/or Se.
[00378] In some embodiments of any one of the aspects described herein, the oligonucleotide or at least one (e.g., both) strand of a dsRNA described herein comprises a 5’-vinylphosphonate group. For example, 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. In some other non-limiting example, the oligonucleotide comprises a 5’-Z- vinylphosphonate group.
[00379] In one example, the 5 ’-modification can be placed in the antisense strand of a double- stranded nucleic acid, e.g., dsRNA molecule. For example, the antisense comprises a 5’-E- vinylphosphonate. In some other non-limiting example, the antisense strand comprises a 5’-Z- vinylphosphonate group.
[00380] In another example, the 5 ’-terminal nucleotide of the antisense strand comprises a 5’- cyclopropylphosphonate group, that is, a group of the formula or a salt thereof,
Figure imgf000101_0001
that is connected to the 4’-C of the 5 ’-end nucleotide.
[00381] In another example, the 5 ’-terminal nucleotide of the antisense strand comprises a group of the formula wherein each Rpp is independently hydrogen or a Cl-6alkyl
Figure imgf000101_0002
(e.g., methyl) or a salt thereof, connected to the 4’-C of the 5’-terminal nucleotide. In one example, the group is of the formula
Figure imgf000102_0001
or a salt thereof.
[00382] In some embodiments, 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. In some embodiments, the 5 ’-terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphodiester linkage to an abasic nucleotide. In some embodiments, , the 5’- terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphorothioate linkage to an abasic nucleotide.
[00383] In some embodiments, the sense strand comprises an inverted abasic acid modification at the 3 ’-end. In some embodiments, the 3 ’-terminal nucleotide of the sense strand comprises a 3’- >3’ phosphodiester linkage to an abasic nucleotide. In some embodiments, he 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphorothioate linkage to an abasic nucleotide. [00384] In some embodiments, 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.
[00385] In some embodiments, 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. In some embodiments, 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.
[00386] In some embodiments, 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.
[00387] In each of the preceding embodiments of a terminal modification by 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. Without wishing to be bound by a theory, 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. Accordingly, in some embodiments, 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. In some embodiments, 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. In some further embodiments, 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.
[00389] In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand. In some embodiments, 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. For instance, 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.
[00390] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16 from the 5 ’-end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14 and 16 from the 5’-end. In still some other embodiments, the antisense comprises stabilizing modifications at positions 2, 14 and 16 from the 5 ’-end.
[00391] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, 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. In some embodiments, 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. [00392] In some embodiments, 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. In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, 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. In some embodiments, 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.
[00393] In some embodiments, 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.
[00394] It is noted a thermally stabilizing modification can replace a 2’-fluoro nucleotide in the sense and/or antisense strand. For example, 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. Similarly, a 2’-fluoro nucleotide at position 14, counting from 5’-end, of the antisense strand, can be replaced with a thermally stabilizing modification.
[00395] For the dsRNA molecules to be more effective in vivo, 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. 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 6 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 7 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 8 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 9 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 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. 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 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. 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 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.
Uses of oligonucleotides and dsRNAs
[00396] In some embodiments of any one of the aspects, 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.
[00397] Accordingly, in another aspect, the disclosure is directed to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene. In some embodiments, 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.
[00398] In another aspect, 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] In some embodiments, the oligonucleotide and/or dsRNA molecule described herein is administered in buffer.
[00400] In some embodiments, 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. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the siRNA is in an aqueous phase, e.g., in a solution that includes water.
[00401] 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). Generally, the siRNA composition is formulated in a manner that is compatible with the intended method of administration, as described herein. For example, in particular embodiments 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.
[00402] 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. Still other agents include chelating agents, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
[00403] In some embodiments, 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. 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.
[00404] In some embodiments, the oligonucleotide and/or dsRNA preparation includes at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA). For example, a oligonucleotide and/or dsRNA composition for the treatment of a viral disease, e.g., HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a dsRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.
[00405] Exemplary formulations which can be used for administering the oligonucleotide and/or dsRNA according to the present invention are discussed below. [00406] Liposomes. A oligonucleotide and/or dsRNA preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “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. As the merging of the liposome and cell progresses, 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. In some embodiments, the liposomes are also specifically targeted, e.g., to direct the oligonucleotide and/or dsRNA to particular cell types.
[00407] A liposome containing oligonucleotide and/or dsRNA can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, 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.
[00408] If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, 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.
[00409] Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate apolynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. 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. Acta 557:9, 1979; Szoka, etal. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984, which are incorporated by reference in their entirety. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, which is incorporated by reference in its entirety). 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.
[00410] 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).
[00411] One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral liposome compositions, for example, 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). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
[00412] Examples of other methods to introduce liposomes into cells in vitro and 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.
[00413] In some embodiments, 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.
[00414] Further advantages of liposomes include: 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.
[00415] A positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) 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).
[00416] A DOTMA analogue, l,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ 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. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, l,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moi eties are linked by ester, rather than ether linkages.
[00417] Other reported 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”) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5 -carboxy spermyl-ami de (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
[00418] 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). For certain cell lines, 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). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
[00419] Liposomal formulations 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. In some implementations, 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. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Lould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987, which are incorporated by reference in their entirety).
[00420] 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.
[00421] 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. Lor example, 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. 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. 61/018,616, fded January 2, 2008; 61/018,611, fded January 2, 2008; 61/039,748, filed March 26, 2008; 61/047,087, filed April 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed October 3, 2007 also describes formulations that are amenable to the present invention.
[00423] Surfactants. The oligonucleotide and/or dsRNA compositions can include a surfactant. In some embodiments, the dsRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, NY, 1988, p. 285).
[00424] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. 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.
[00425] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. 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.
[00426] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
[00427] If 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. [00428] 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).
[00429] Micelles and other Membranous Formulations. “Micelles” 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.
[00430] 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 provide smaller size micelles.
[00431] In one method, a first micellar composition is prepared 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. In another method, 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.
[00432] Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, 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.
[00433] 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.
[00434] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1, 1,2 tetrafluoroethane) may be used.
[00435] 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.
[00436] Particles. In some embodiments, 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.
Pharmaceutical compositions
[00437] 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.
[00438] The pharmaceutical 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.
[00439] The phrase “therapeutically-effective amount” as used herein 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.
[00440] The phrase “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.
[00441] The phrase “pharmaceutically-acceptable carrier” as used herein 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. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of 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; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
[00442] 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.
[00443] In certain embodiments, 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. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound of the present invention.
[00444] 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. In general, 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.
[00445] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally- administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
[00446] The 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.
[00447] The term “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.
[00448] 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.
[00449] 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.
[00451] In one aspect, provided herein is a method of administering an oligonucleotide and/or dsRNA described herein, to a subject (e.g., a human subject). In another aspect, 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. In some embodiments, 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 1016 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.
[00452] 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. In some embodiments dosages may be less than 10, 5, 2, 1, or 0.1 mg/kg of body weight.
[00453] In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.
[00454] In some embodiments, the effective dose is administered with other traditional therapeutic modalities.
[00455] In some embodiments, 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. In certain embodiments 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. Following treatment, 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.
[00456] 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.
[00457] In some embodiments, the composition includes a plurality of dsRNA molecule species. In another embodiment, the dsRNA molecule species has sequences that are non- overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of dsRNA molecule species is specific for different naturally occurring target genes. In another embodiment, the dsRNA molecule is allele specific. [00458] The oligonucleotide and/or dsRNA described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.
[00459] In some embodiments, 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.
[00460] The invention provides methods, compositions, and kits, for rectal administration or delivery of oligonucleotide and/or dsRNA composition described herein.
Methods of inhibiting expression of a target gene
[00461] 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. [00462] 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.
[00463] Another aspect 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. In some embodiments, 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 gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAFl/CIPl) gene, mutations in the p27(KIPl) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MT Al gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.
[00464] Some exemplary aspects of the disclosure are described by one or more of following numbered Embodiments:
[00465] Embodiment 1: An oligonucleotide comprising at least one nucleoside (e.g., one) of
Figure imgf000118_0001
Formula (IV): Formula (IV) , wherein: jy js an optionally substituted nucleobase; XM is CEE,
O, NRN or S (where RN 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); R43 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 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(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or a nitrogen protecting group; R45 represents a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker covalently bonded 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-methoxy ethyl), alkoxy alkylamine, alkoxy oxy carboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl-ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P- 0-5'), monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates [(Rp)(0H)(0)P- 0-5', Rp is optionally substituted C1-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(Rpl)(0H)(0)P-0-5', RP1 is alkoxyalkyl, e.g., methoxymethyl (CEEOMe) or ethoxymethyl ], (HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a- P(X)(0H)-0]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[- (CH2)a-0-P(X)(0H)-0]b- 5', H[-(CH2)a-0-P(X)(0H)-0]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H0[-(CH2)a-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[-(CH2)a-P(X)(0H)-0]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', wherein X is O or S, a and b are each independently 1-10, or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group (e.g., -CH=CH-XP, Xp is a phosphonate group), C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), provided that only one of R43 and R45 is a solid support or linker covalently bonded to a solid support; and each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl, and provided that (i) when R43 is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R45 is a bond to an intemucleotide linkage to a preceding nucleotide; and (ii) when R45 is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R43 is a bond to an intemucleotide linkage to a preceding nucleotide. [00466] Embodiment 2: The oligonucleotide of Embodiment 1, wherein R43 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. [00467] Embodiment 3: The oligonucleotide of Embodiment 2, wherein R43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
[00468] Embodiment 4: The oligonucleotide of Embodiment 2, wherein R43 is a solid support, or a linker (e.g., -C(O)CH2CH2C(O)-) covalently bonded to a solid support.
[00469] Embodiment 5: The oligonucleotide of Embodiment 2, wherein R43 is either (i) hydrogen or a nitrogen protecting group; or (ii) hydroxyl or a protected hydroxyl.
[00470] Embodiment 6: The oligonucleotide of any one of Embodiments 1-5, wherein R45 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 R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
[00471] Embodiment 7: The oligonucleotide of Embodiment 6, wherein R45 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 R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
[00472] Embodiment 8: The oligonucleotide of Embodiment 7, wherein R45 is a bond to an intemucleotide linkage to a preceding nucleotide.
[00473] Embodiment 9: The oligonucleotide of Embodiment 7, wherein R45 is hydroxylprotected hydroxyl, or optionally substituted C1-30 alkoxy, or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) (E-vinylphosphonate) group..
[00474] Embodiment 10a: The oligonucleotide of any one of Embodiments 1-9, wherein XM is CEE.
[00475] Embodiment 10b: The oligonucleotide of any one of Embodiments 1-9, wherein XM is O.
[00476] Embodiment 10c: The oligonucleotide of any one of Embodiments 1-9, wherein XM is S.
[00477] 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.
[00478] Embodiment 12: The oligonucleotide of any one of Embodiments 1-11, wherein the oligonucleotide comprises from 3 to 50 nucleotides.
[00479] Embodiment 13: The oligonucleotide of any one of Embodiments 1-12, wherein the oligonucleotide comprises at least one ribonucleotide.
[00480] Embodiment 14: The oligonucleotide of any one of Embodiments 1-13, wherein the oligonucleotide comprises at least one 2’-deoxyribonucleotide.
[00481] 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).
[00482] 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).
[00483] 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).
[00484] 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).
[00485] 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).
[00486] 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).
[00487] Embodiment 21: The oligonucleotide of any one of Embodiments 1-20, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.
[00488] Embodiment 22: The oligonucleotide of any one of Embodiments 1-21, wherein the oligonucleotide is attached to a solid support.
[00489] Embodiment 23: The oligonucleotide of any one of Embodiments 1-22, wherein oligonucleotide comprises at least one ligand.
[00490] Embodiment 24: The oligonucleotide of any one of Embodiments 1-23, wherein the oligonucleotide comprises at least one hydroxyl, phosphate or amino protecting group. [00491] 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.
[00492] 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.
[00493] 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.
[00494] 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.
[00495] Embodiment 29: The double-stranded nucleic acid of Embodiment 28, wherein the first and second strand are independently 15 to 25 nucleotides in length.
[00496] Embodiment 30: The double-stranded nucleic acid any one of Embodiments 28-29, wherein double-stranded nucleic acid is capable of inducing RNA interference.
[00497] 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.
[00498] 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.
[00499] 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.
[00500] 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.
Figure imgf000122_0001
[00501] Embodiment 35: A compound of Formula (III): Formula (III) , wherein: jy js an optionally substituted nucleobase; XM is CEE, O, NRN or S (where RN 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); R33 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)-) 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 C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl- ON(CH2R8)(CH2R9), or -O-C4-30alkyl-ON(CH2R8)(CH2R9); R35 is hydroxy, protected hydroxy, phosphate group, a reactive phosphorous group, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R8)(CH2R9), -O-C4-30alkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P- O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates (R(OH)(O)P-O- 5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc…), alkyletherphosphonates (R(OH)(O)P-O- 5', R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc…), (HO)2(X)P-O[-(CH2)a-O- P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O- P(X)(OH)-O]b- 5', where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[-(CH2)a-O-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[-(CH2)a-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', wherein X is O or S; and a and b are each independently 1-10), or R35 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group or C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate), provided that only one of R33 and R35 is a reactive phosphorous group; and each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30alkynyl. [00502] Embodiment 36: The compound of Embodiment 35, wherein R33 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. [00503] Embodiment 37: The compound of Embodiment 35, wherein R33 is a H or nitrogen protecting group.
[00504] Embodiment 38: The compound of any one of Embodiments 35-37, wherein R35 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 R35 taken together with the carbon to which it is attached form avinylphosphonate group.
[00505] Embodiment 39: The compound of any one of Embodiments 35-28, wherein R35 is a reactive phosphorous group, linker, solid support, a linker covalently bonded to a solid support, hydroxyl, or a protected hydroxyl.
[00506] Embodiment 40: The compound of any one of Embodiments 35-39, wherein R35 is reactive phosphorous group, solid support, or a linker covalently bonded to a solid support.
[00507] Embodiment 41: The compound of any one of Embodiments 35-40, wherein R35 is - P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); and RP4 is halogen (e.g., Cl)
[00508] Embodiment 42: The compound of any one of Embodiments 35-41, wherein XM is CEE.
[00509] Embodiment 43a: The compound of any one of Embodiments 35-41, wherein XM is O.
[00510] Embodiment 43b: The compound of any one of Embodiments 35-41, wherein XM is S.
[00511] Embodiment 44: The compound of Embodiment 35, wherein: XM is CEE; R33 is H or nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., - P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); andRP4 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.
[00512] Embodiment 45: The compound of Embodiment 35, wherein:XM is O; R33 is H or nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., - P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); and RP4 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.
[00513] Embodiment 46: The compound of Embodiment 35, wherein:XM is S; R33 is H or nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., - P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci- Cealkyl (e.g., methyl); and RP4 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.
[00514] Embodiment 47: The compound of Embodiment 35, wherein: XM is CEE; R35 is hydroxyl or a protected hydroxyl; and R33 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)- RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.
[00515] Embodiment 48: The compound of Embodiment 47, wherein XM is CEE; R35 is hydroxyl or a protected hydroxyl; and R33 is a solid support or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.
[00516] Embodiment 49: The compound of Embodiment 35, wherein: XM is O; R35 is hydroxyl or protected hydroxyl; and R33 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); andRP4 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.
[00517] Embodiment 50: The compound of Embodiment 49, wherein: XM is O; R35 is hydroxyl or protected hydroxyl; and R33 is a solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.
[00518] Embodiment 51: The compound of Embodiment 35, wherein: XM is S; R35 is hydroxyl or protected hydroxyl; and R33 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); andRP4 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.
[00519] Embodiment 52: The compound of Embodiment 51, wherein: XM is O; R35 is hydroxyl or protected hydroxyl; and R33 is a solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.
[00520] 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. [00521] Embodiment 54: The compound of any one of Embodiments 35-53, wherein B and B’ are independently adenine, cytosine, guanine, thymine, uracil,
Figure imgf000126_0001
Figure imgf000126_0002
Figure imgf000127_0001
selected from 1 to 10; and R1 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.
[00522] Embodiment 55: A compound selected from the group consisting of:
Figure imgf000127_0002
Figure imgf000128_0001
Figure imgf000129_0001
[00523] Embodiment 56: An oligonucleotide prepared using a compound of any one of
Embodiments 35-55.
[00524] 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.
[00525] 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.
[00526] 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.
[00527] 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.
[00528] 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.
[00529] 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.
[00530] 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.
[00531] 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).
[00532] 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).
[00533] 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).
[00534] 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).
[00535] 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).
[00536] 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).
[00537] 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.
[00538] 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).
[00539] 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).
[00540] 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.
[00541] 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.
[00542] 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.
[00543] 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.
Some selected definitions
[00544] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
[00545] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.
[00546] Further, 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. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001). [00547] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[00548] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
[00549] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
[00550] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[00551] As used herein, the term “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. The 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. Exemplary 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.
[00552] 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. In certain embodiments, the heteroatom(s) is placed at any interior position of the heteroalkyl group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, -CH2- CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2- CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-0-CH3, -SI(CH3)3, -CH2-CH=N- OCH3, and -CH=CH-N(CH3)-CH3. In some embodiments, up to two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3
[00553] As used herein, the term “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.
[00554] As used herein, the term “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.
[00555] As used herein, the term “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.
[00556] “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 Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 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.
[00557] “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. Exemplary aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl.
[00558] “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.
[00559] 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, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-l,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4- oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H- pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can be substituted by a substituent. [00560] As used herein, the term “halogen” or “halo” refers to an atom selected from fluorine, chlorine, bromine and iodine. The term “halogen radioisotope” or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.
[00561] A “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.
[00562] The term “haloalkyl” as used herein 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. The terms “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).
[00563] As used herein, the term “amino” means -NHz. The term “alkylamino” means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., -NH(alkyl). The term “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). The term “alkylamino” includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.” The term “arylamino” means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl, and — N(aryl)z. The term “heteroarylamino” means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example — NHheteroaryl, and — N(heteroaryl)2. Optionally, two substituents together with the nitrogen can also form a ring. Unless indicated otherwise, 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(CH3)2. Exemplary dialkylamino includes, but is not limited to, — N(Ci-Cioalkyl)2, such as N(CH3)2, — N(CH2CH3)2, — N(CH2CH2CH3)2, and — N(CH(CH3)2)2.
[00564] The term “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. For example, 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.
[00565] The terms “hydroxyl” and “hydroxyl” mean the radical — OH.
[00566] The terms “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.
[00567] As used herein, the term “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.
[00568] As used herein, the term “oxo” means double bonded oxygen, i.e., =0.
[00569] The term “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.
[00570] The term “ester” refers to a chemical moiety with formula -C(=O)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl.
[00571] The term “cyano” means the radical — CN.
[00572] The term “nitro” means the radical — NO2.
[00573] The term, “heteroatom” refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens. A “heteroatom moiety” includes a moiety where the atom by which the moiety is attached is not a carbon. Examples of heteroatom moieties include — N=, — NRN — , — N (O )=, — O — , — S — or — S(O)2 — , — OS(O)2 — , and — SS — , wherein RN is H or a further substituent.
[00574] The terms “alkylthio” and “thioalkoxy” refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur. In preferred embodiments, the “alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl. Representative alkylthio groups include methylthio, ethylthio, and the like. The term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups. “Arylthio” refers to aryl or heteroaryl groups.
[00575] The term “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.
[00576] The term “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.
[00577] The term “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.
[00578] “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.
[00579] “Aroyl” means an aryl-CO — group, wherein aryl is as previously described. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.
[00580] “Arylthio” refers to an aryl-S — group, wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.
[00581] “Aralkyl” refers to an aryl-alkyl — group, wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
[00582] “Aralkyloxy” refers to an aralkyl-0 — group, wherein the aralkyl group is as previously described. An exemplary aralkyloxy group is benzyloxy.
[00583] “Aralkylthio” refers to an aralkyl-S — group, wherein the aralkyl group is as previously described. An exemplary aralkylthio group is benzylthio.
[00584] “Alkoxycarbonyl” refers to an alkyl-0 — CO — group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
[00585] “Aryloxycarbonyl” refers to an aryl-0 — CO — group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
[00586] “Aralkoxycarbonyl” refers to an aralkyl-0 — CO — group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
[00587] “Carbamoyl” refers to an H2N — CO — group.
[00588] “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. [00589] “Dialkylcarbamoyl” refers to R'RN — CO — group, wherein each of R and R' is independently alkyl as previously described.
[00590] “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.
[00591] The term “optionally 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. The term “substituents” 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. In some cases, two substituents, together with the carbons to which they are attached to can form a ring.
[00592] For example, any alkyl, alkenyl, cycloalkyl, heterocyclyl, heteroaryl or aryl is optionally substituted with 1, 2, 3, 4 or 5 groups selected from OH, CN, -SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci- C4)alkyl, N[(Ci-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (Ci-Cs)alkyl, O(Ci-C8)alkyl, O(Ci- Cs)haloalkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(O)- alkyl, C(O)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m — (CH2)P — OH, CH2 — [CH(OH)]m — (CH2)P — NH2or CH2-aryl-alkoxy; “m” and “p” are independently 1, 2, 3, 4, 5 or 6.
[00593] In some embodiments, 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.
[00594] An “isocyanato” group refers to a NCO group.
[00595] A “thiocyanato” group refers to a CNS group. [00596] An “isothiocyanate” group refers to a NCS group.
[00597] “Alkoyloxy” refers to a RC(=O)O- group.
[00598] “Alkoyl” refers to a RC(=O)- group.
[00599] As used herein, the terms “dsRNA”, “siRNA”, and “iRNA agent” are used interchangeably to refer to agents that can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene, exogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.
[00600] As used herein, the phrase “mediates 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.
[00601] By “specifically hybridizable” and "complementary" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non- traditional types. In reference to the nucleic molecules of the present invention, 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. USA 83:9373-9377; Turner et al., 1987, /. Am. Chem. Soc. 109:3783-3785). 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. Less than perfect complementarity refers to the situation in which some, but not all, 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. [00602] 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. For example, 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.
[00603] As used herein, the term “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.
[00604] As used herein, the term “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.
[00605] As used herein, the term “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).
[00606] As used herein, unless otherwise indicated, the term “methyleneoxy LNA” alone refers to P-D-methyleneoxy LNA.
[00607] As used herein, the term “MOE” refers to a 2'-O-methoxyethyl substituent.
[00608] As used herein, 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. Such modifications include nucleobase, monomeric linkage, and sugar modifications as well as the absence of modification (unmodified). Thus, in certain embodiments, the nucleotide linkages in each of the wings are different than the nucleotide linkages in the gap. In certain embodiments, each wing comprises nucleotides with high affinity modifications and the gap comprises nucleotides that do not comprise that modification. In certain embodiments the 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. In certain embodiments, the modifications in the wings are the same as one another. In certain embodiments, the modifications in the wings are different from each other. In certain embodiments, nucleotides in the gap are unmodified and nucleotides in the wings are modified. In certain embodiments, the modification(s) in each wing are the same. In certain embodiments, the modification(s) in one wing are different from the modification(s) in the other wing. In certain embodiments, oligomeric compounds are gapmers having 2'-deoxynucleotides in the gap and nucleotides with high-affinity modifications in the wing.
[00609] The term “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). Examples of BNA nucleotides include the following nucleosides:
Figure imgf000141_0001
oxyamino BNA vinyl-carbo BNA
[00610] The term ‘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:
Figure imgf000141_0002
[00611] The term ‘ENA’ refers to ethylene-bridged nucleic acid, and is often referred as constrained or inaccessible RNA.
[00612] 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. And the target cleavage site region comprises at least one or at least two nucleotides on both side of the cleavage site. For the sense strand, 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. As is well understood in the art, 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.
[00613] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about
10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about
40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about
65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 98%, at least about 99% or more. As used herein, “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.
[00614] As used herein, 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. For example, 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. Similarly, 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.
[00615] For example, 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.
[00616] Similarly, 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.
[00617] As used herein, 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. For example, 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.
[00618] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g. animal or a plant). As used herein, the term “ex vivo” refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube). As used herein, the term "in vivo" refers to events that occur within an organism (e.g. animal, plant, and/or microbe).
[00619] As used herein, 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. Usually 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. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female. [00620] Preferably, 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. In addition, compounds, compositions and methods described herein can be used to with domesticated animals and/or pets.
[00621] In some embodiments, the subject is human. In another embodiment, 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. In some embodiments, the subject can be of European ancestry. In some embodiments, the subject can be of African American ancestry. In some embodiments, the subject can be of Asian ancestry.
[00622] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will self-administer by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.
[00623] As used herein, the term “parenteral administration,” refers to administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
[00624] As used herein, the term “subcutaneous administration” refers to administration just below the skin. “Intravenous administration” means administration into a vein.
[00625] As used herein, the term “dose” refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.
[00626] As used herein, the term “dosage unit” refers to a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial comprising lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
[00627] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., provided herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. The invention is further illustrated by the following example, which should not be construed as further limiting.
EXAMPLES
EXAMPLE 1: Synthesis and Biophysical Properties of Carbocyclic Morpholino-containing Oligonucleotides
[00628] In the following example, applicants report the synthesis of carbocyclic morpholino nucleotide phosphoramidates and their incorporation into phosphorodiamidate morpholino oligonucleotides (PMOs). A PMO containing carPMO residues formed duplexes with both DNA and RNA, and car-PMO oligomer had higher stability at endosomolytic pH and higher hydrophobicity than PMO Oligomer.
[00629] Phosphorodiamidate morpholino oligonucleotides (PMOs)1 and carbocyclic nucleosides (car-nucleosides) were developed for use in oligonucleotide therapeutics.2 To date, four 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 viral4a and bacterial infections411 and cancers5 in cell-based and preclinical models. In a PMO monomer, the five-membered ribose sugar unit of a natural nucleotide is replaced by a morpholino ring (Figures 8A-8B), whereas in a car-nucleoside the furanose ring oxygen is replaced by a CH2 group. These sugar modifications enhance resistance against nuclease degradation and stabilize the glycosidic bond against hydrolytic cleavage. Several car-nucleosides have been approved as antitumor and antiviral therapies.6
[00630] 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.8 Recently, Borbas’s group reported alkylamine-linked, cationic morpholino oligonucleotide analogues.11 [00631] Here we report synthesis of carbocyclic PMOs (carPMOs) (Figures 8A-8B). The four car-morpholino nucleoside monomers, car-A, car-U, car-G, and car-C, and corresponding chlorophosphoramidate monomers were prepared (Figure 8A). These were incorporated into PMOs in order to evaluate the stability of their duplexes with DNA and RNA as well as their conformational properties using CD spectroscopy.
[00632] To incorporate carPMO into the PMO backbone, 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. Using (NH^I^ChM^O as a nitrogen source following the original procedure,12 7 was obtained. After A-tritylation, 5 was obtained. But in our hands the yield was poor. Alternatively, hydrogenolysis of the benzyl group and subsequent N- tritylation of 7 afforded 5 in 41.5% yield from 3, which was then treated with TBAF to obtain protected car-U morpholino monomer 8. The conformations of 5 and its morpholino analogue are similar as demonstrated by X-ray crystallographic analyses (Figure 11).
Figure imgf000146_0001
Scheme 1. Synthesis of car-U morpholino monomer 8"
"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. [00633] To convert U to C, the general procedure using the triazole derivative was followed (Scheme 2).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.
Figure imgf000147_0001
Scheme 2. Synthesis of car-C morpholino monomer 11"
Synthesis of car-C morpholino monomer 11. ® Reagents and conditions: (i) (a) 1,2,4-triazole, POCk, EtsN, CH3CN, THF, 0 °C to room temperature, (b) ammonium hydroxide, THF, room temperature; (ii) AC2O, DMAP, pyridine, room temperature; (iii) TBAF, THF, room temperature.
[00634] For the synthesis of car-A morpholino monomer,20 the carbocyclic moiety was introduced into the aminopyrimidine ring through a nucleophilic substitution reaction with 5- amino-4,6-dichloropyrimidine, resulting in selective replacement of one chlorine to yield 12. The 5-amino-6-chloropyrimidine was converted to the 6-chloropurine ring via construction of the imidazole ring through a reaction with triethylorthoformate to provide 13.
Figure imgf000148_0001
Scheme 3: Synthesis of car-A morpholino monomer 21"
"Reagents and conditions: (i) 5-amino-4,6-dichloropyrimidine, EtsN, M-BUOH, reflux; (ii) (EtO)3CH, cone. HC1, room temperature; steps iii, iv, v as per Scheme 1; (vi) NH4OH, 1,4-dioxane, MW, 100 °C; (vii) benzoyl chloride, pyridine, 0 °C to room temperature; (viii) 10% Pd on carbon, HCO2H, EtOH, reflux; (ix) trityl chloride, EtsN, DMF, room temperature; (x) TBAF, THF, room temperature. Bz = benzoyl.
[00635] The hydroxyl group of 13 was protected with TBDPS to yield 14. Dihydroxylation of the olefin afforded the diol 15 as a mixture of lyxo and ribo isomers that were then converted to N- benzylmorpholino 16 as per Scheme 1. The adenine ring was then obtained by treatment of 16 with NH4OH solution. Next, the exocyclic amine of 17 was protected with the benzoyl group to obtain 18. For hydrogenation of 18, various conditions were evaluated because loss of the A-benzyl group was observed (Table 2). The desired product 19 was eventually obtained in 55% yield. Subsequent A-protection with a trityl group, followed by treatment with TBAF provided car-A morpholino monomer 21 in 11% yield from 1 (Scheme 3).
[00636] For the synthesis of the car-G morpholino monomer,20 we constructed the 2-amino-6- chloropurine ring through the condensation of aminocarbocycle 1 and 2-amino-4,6-dichloro-5- formamido-pyrimidine to obtain 2-amino-6-chloropurine carbocycle 22 (Scheme 4). Silyl- protection of the OH yielded compound 23, which was treated with the sodium salt of 3- hydroxypropionitrile, converting the amino-chloropurine to the guanine ring of 24. The exocyclic amine was protected with z.so-butryl amide to provide compound 25. Oxidative cleavage and subsequent reductive amination gave the A-benzyl carbocyclic morpholino 27. The benzyl protection was removed by Pd-catalyzed hydrogenation to obtain the secondary amine 28, which was then protected with trityl to yield the fully protected intermediate 29. Subsequent deprotection of the TBDPS group gave the trityl-protected car-G morpholino monomer 30 in 17% yield from 1. Crystal structures of the various monomer intermediates can be found in Figure 9 (Figure 11 for enlarged overlaid structures) which confirm configurational and structural features of monomers described.
Figure imgf000149_0001
Scheme 4. Synthesis of car-G morpholino monomer 30"
"Reagents and conditions: (i) 2-amino-4,6-dichloro-5-formamidopyrimidine, DIPEA, EtOH, H2O, MW, 140 °C; (ii) TBDPSC1, imidazole, DMF, room temperature; (iii) 3 -hydroxypropionitrile, NaH, THF, 0 °C to room temperature; (iv) isobutyryl chloride, pyridine, 0 °C to room temperature; Steps v and vi as per Scheme 1; (vii) 10% Pd on carbon, AcOH, 1,3 -cyclohexadiene, EtOH, reflux; (viii) trityl chloride, EtsN, DMF, room temperature; (ix) TBAF, THF, room temperatur.e. z-Bu = isobutyryl.]
[00637] With these monomers in hand, we synthesized the chlorophosphoramidate monomers necessary for the synthesis of carPMO (Table 1). Our initial attempts to synthesize the chloro phosphorami date monomers using LiBr/DBU13a or ETT methods 13b were not successful, with products obtained in less than 10% yield. We therefore performed the reaction in the presence of strong base. The reaction was carried out in ice-salt mixture as we found that the product decomposed otherwise. Although the reaction worked well in the presence of LiHMDS for regular morpholino monomers as reported earlier,14 use of LDA resulted in better yields than LiHMDS. At -78 °C in LDA, the reaction was very slow and yield was approximately 20%. By comparison, under ice-salt conditions, the yield was 51% with a side product having an additional attachment of chlorophosphoramidate to C=O of U isolated in 10% yield.
Table 1: Synthesis of chlorophosphorodiamidate car-morpholino monomers"
Figure imgf000150_0001
Car-morpholino monomers Chlorophosphoramidates
Figure imgf000150_0002
"Conditions: car morpholino monomer (1 equiv), base (2.5 equiv), A,A-dimethylphosphoramic di chloride (3 equiv) in dry THF at ice-salt temperature. Time, 30 min. bReaction was performed at -78°C.
[00638] In summary, we synthesized carbocyclic morpholino monomer building blocks and incorporated these residues into the PMO backbone to generate PMO-carPMO chimeras. Oligomers were synthesized on solid support using a combination of manual and automated synthesis. The car-morpholino chlorophosphoramidate monomers were incorporated manually to use lesser reagent than when coupling was automated. The stabilities of duplexes between DNA and PMO-carPMO chimeras was lower than that of the DNA:PMO duplex. Incorporation of one or two car-A, car-U, or car-C residues into PMO resulted in either comparable or higher duplex stability with RNA; however, multiple car-morpholino monomers destabilized the hybrid duplexes. It is important to mention that all the modified chimeric PMO-carPMOs can form duplexes. Whilst high melting temperatures of duplexes with targets is one requirement for therapeutic applications of oligonucleotide-based drugs, other parameters such as metabolic stability, cell permeation, release from the endolysosomal compartments, and target specificity are also critical.
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Synthetic procedures and characterization data for the new compounds
General conditions
[00639] 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. All commercially available reagents and solvents were purchased from Sigma-Aldrich unless otherwise stated and were used as received. 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. 13C NMR spectra were recorded at 75, 101, 126, or 151 MHz. 31P NMR were recorded at 121 MHz. Chemical shifts are given in ppm referenced to the solvent residual peak (DMSO-c/e - 'H: 8 at 2.50 ppm and 13C 8 at 39.5 ppm, CDCk -1H: 8 at 7.26 ppm, and 13C 8 at 77.133 ppm, CD2CI2 - 'H: 8 at 5.32 ppm, and 13C 8 at 53.5 ppm, CD3OD - 'H: 8 at 3.35, 4.78 ppm, and 13C 8 at 49.3 ppm. Coupling constants are given in Hz. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), septet (sept), broad signal (brs), or multiplet (m).
Synthesis of compound 3
Figure imgf000152_0001
2 3
[00552] To a solution of compound 2 prepared as described1 (5.4 g, 25.9 mmol) and imidazole (5.30 g, 77.8 mmol) in DMF (100 mL) was added Zert-butyldiphenylsilyl chloride (10.7 g, 38.9 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was quenched with saturated NaHCO3 (aq.) and diluted with diethyl ether. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–50% ethyl acetate in hexanes) to obtain compound 3 as a white solid (10.9 g, 94%). [00553] 1H NMR (500 MHz, DMSO-d6) 511.24 (s, 1H), 7.40–7.60 (m, 10H), 7.24 (d, J = 8.1 Hz, 1H), 6.11 (ddd, J =2.2, 2.2 and 5.6 Hz, 1H), 5.73 (ddd, J =2.2, 2.2 and 5.6 Hz, 1H), 5.46–5.51 (m, 1H), 5.45–5.49 (m, 1H), 5.34 (dd, J = 2.2 and 8.0 Hz, 1H), 3.63–3.71 (m, 2H), 2.91–2.93 (m, 1H), 2.47–2.53 (m, 1H), 1.33 (ddd, J = 6.6, 6.6 and 13.8 Hz, 1H), 0.99 (s, 9H). 13C NMR (126 MHz, DMSO-d6) 5 163.2, 150.8, 141.1, 138.6, 135.1, 135.0, 133.0, 132.9, 130.0, 129.9, 129.8, 127.9, 127.8, 101.4, 65.9, 60.6, 46.8, 32.9, 26.7, 18.8. HRMS (ESI) m/z: [M + H]+ calc. for C26H31N2O3Si 447.2104; found 447.2102. Synthesis of compound 4
Figure imgf000153_0001
[00554] Compound 3 (5 g, 11.2 mmol) was dissolved in a 5:1 acetone-water mixture (60 mL), and then osmium tetroxide (4 wt% in H2O; 3 mol%) and N-methylmorpholine-N-oxide (3.28 g, 28.0 mmol) were added to the solution. The mixture was stirred at room temperature for 24 h, and the reaction quenched with saturated Na2S2O3 (aq.), and stirred for 30 min. The reaction mixture was diluted in ethyl acetate, and the aqueous layer was extracted with ethyl acetate. The combined organic layer was dried (Na2SO4) and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (65–100% ethyl acetate in hexanes) to obtain compound 4 as a white foam (5.22 g, 97%). [00555] 1H NMR (600 MHz, DMSO-d6) δ 11.24 (d, J =2.3 Hz, 0.5H), 11.14 (d, J =2.3 Hz, 0.5H), 7.77 (d, J = 8.1 Hz, 0.5H), 7.60–7.65 (m, 4.5H), 7.43–7.47 (m, 6H), 5.56 (dd, J =2.3 and 8.1 Hz, 0.5H), 5.51 (dd, J =2.3 and 8.1 Hz, 0.5H), 5.15 (d, J = 5.2 Hz, 0.5H), 4.95–5.03 (m, 1.5H), 4.66–4.67 (m, 0.5H),4.57–4.61 (m, 0.5H), 3.99–4.08 (m, 1.5H),3.91–3.94 (m, 0.5H),3.83–3.84 (m, 0.5H),3.62–3.72 (m, 1.5H),2.01–2.13 (m, 2H),1.44–1.49 (m, 0.5H), 1.35–1.49 (m, 0.5H),0.99–1.01 (m, 9H). 13C NMR (151 MHz, DMSO-d6) δ 163.80, 163.69, 152.12, 151.76, 145.99, 143.39, 135.55, 135.52, 135.51, 133.81, 133.77, 133.61, 133.58, 130.32, 130.25, 130.21, 128.38, 128.34, 128.31, 101.57, 100.13, 73.42, 71.69, 71.46, 71.36, 65.68, 63.86, 61.22, 53.70, 45.25, 41.85, 32.62, 27.96, 27.16, 27.14, 19.36, 19.30. HRMS calc. for C26H33N2O5Si [M + H]+ 481.2159, found 481.2152. Synthesis of compound 5
Figure imgf000154_0001
[00556] Compound 5 was synthesized following a described procedure for PMO compound previously.2,3 To a solution of compound 4 (5 g, 10.4 mmol) in MeOH (50 mL), sodium periodate (2.45 g, 11.4 mmol) and ammonium biborate tetrahydrate (3.01 g, 11.4 mmol) were added. The mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC until disappearance of compound 4. The filtrate was recovered after filtration through a pad of celite. Sodium cyanoborohydride (1.44 g, 22.9 mmol) was added dropwise to the filtrate and molecular sieves (4 Å, 300 mg/mmol) were added, followed by the dropwise addition of acetic acid (1.31 mL, 22.9 mmol). The mixture was stirred at room temperature for 12 h and then filtered through a pad of celite. The volatile organic solvents were removed by evaporation. The residue was partitioned between saturated NaHCO3 (aq.) and EtOAc, and the organic layer was washed with water and with brine, dried over Na2SO4, and concentrated under vacuum. The residue was dissolved in DMF (50 mL), and triethylamine (2.90 mL, 20.8 mmol) and trityl chloride (2.90 g, 10.4 mmol) were added to the solution. The mixture was stirred at room temperature for 3 h, and the reaction was quenched with saturated NaHCO3 (aq.) and diluted with diethyl ether. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–40% ethyl acetate in hexanes) to obtain compound 5 as a white solid (610 mg, 8.3%). [00557] 1H NMR (500 MHz, DMSO-d6) δ 11.17 (d, J = 2.3 Hz, 1H), 7.17–7.53 (m, 26H), 5.38 (dd, J = 2.3 and 8.1 Hz, 1H), 4.79–4.86 (m, 1H), 3.59 (dd, J = 4.6 and 10.1 Hz, 1H), 3.41–3.43 (m, 1H), 3.24 (t, J = 10.1 Hz, 1H), 3.11–3.13 (m, 1H), 2.32–2.36 (m, 1H), 1.69–1.72 (m, 1H), 1.20– 1.29 (m, 2H), 0.97 (t, J = 11.0 Hz, 1H), 0.85 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 162.86, 150.76, 141.72, 134.93, 134.91, 132.96, 132.82, 129.73, 129.59, 127.78, 127.74, 127.60, 126.00, 100.91, 76.71, 66.00, 52.51, 52.11, 51.47, 38.52, 30.24, 26.50, 18.67. HRMS calc. for C45H48N3O3Si [M + H]+ 706.3465, found 706.3474. Synthesis of compound 6
Figure imgf000155_0001
[00558] Compound 6 was synthesized essentially as described.4 To a vigorously stirred suspension of chromatographic grade silica gel (20.0 eq.) in MeOH (50 mL) was added dropwise a solution of sodium periodate (2.94 g, 13.7 mmol) in water (5 mL) followed by dropwise addition of a solution of compound 4 (6 g, 12.5 mmol) in MeOH (50 mL). The resulting mixture was stirred at room temperature for 3 h, and the reaction was monitored by TLC until disappearance of 4. To the filtrate obtained after filtration through a pad of celite were added benzylamine (1.50 mL, 13.7 mmol), sodium cyanoborohydride (1.57 g, 25.0 mmol), and molecular sieves (4 Å, 300 mg/mmol), followed by the dropwise addition of acetic acid (1.43 mL, 25.0 mmol). The mixture was stirred at room temperature for 12 h and filtered. The volatile organic solvents were removed by evaporation. The residue was partitioned between saturated NaHCO3 (aq.) and EtOAc, and the organic layer was washed with water and with brine, dried over Na2SO4, and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–70% ethyl acetate in hexanes) to obtain compound 6 as a white foam (4.12 g, 60%). [00559] 1H NMR (400 MHz, DMSO-d6) δ 11.24 (d, J = 2.4 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.52–7.55 (m, 4H),7.38–7.46 (m, 6H), 7.21–7.33 (m, 5H), 5.52 (dd, J = 2.4 and 8.1 Hz, 1H), 4.43– 4.50 (m, 1H), 3.53–3.61 (m, 2H),3.40–3.45 (m, 2H), 2.96–3.00 (m, 1H), 2.84–2.87 (m, 1H), 2.06 (t, J = 10.7 Hz, 1H), 1.90–1.97 (m, 1H), 1.58–1.71 (m, 2H), 1.36 (q, J = 12.2 Hz, 1H), 0.91 (s, 9H). 13C NMR (101 MHz, DMSO-d6) δ 163.31, 150.99, 142.30, 137.93, 135.24, 135.21, 133.21, 133.15, 130.10, 129.08, 128.40, 128.10, 127.24, 101.46, 66.38, 62.00, 56.77, 55.18, 51.57, 38.37, 30.99, 26.80, 18.95. HRMS calc. for C33H40N3O3Si [M + H]+ 554.2839, found 554.2834. Synthesis of compound 7
Figure imgf000156_0001
[00560] To a vigorously stirred suspension of 10% palladium on carbon (300 mg) and ammonium formate (3.42 g, 54.2 mmol) in EtOH (50 mL) was added compound 6 (3 g, 5.42 mmol). The mixture was refluxed overnight and filtered through a pad of celite. The filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–10% MeOH in CH2Cl2) to obtain compound 7 as a white solid (2.16 g, 86%). [00561] 1H NMR (500 MHz, DMSO-d6) δ 7.58–7.63 (m, 5H), 7.40–7.45 (m, 6H), 5.52 (d, J = 7.9 Hz, 1H), 4.29–4.35 (m, 1H), 3.44–3.51 (m, 2H), 3.01 (dd, J = 2.8 and 12.2 Hz, 1H), 2.87 (dd, J = 2.4 and 11.8 Hz, 1H), 2.48–2.52 (m, 1H), 2.13 (t, J = 12.2 Hz, 1H), 1.75–1.89 (m, 2H), 1.42 (q, J = 12.1 Hz, 1H), 0.98 (s, 9H).13C NMR (101 MHz, DMSO-d6) δ 163.00, 150.88, 141.98, 135.00, 133.00, 129.81, 127.86, 101.02, 66.41, 52.25, 49.47, 48.24, 31.81, 26.64, 18.80. HRMS calc. for C26H34N3O3Si [M + H]+ 464.2369, found 464.2364. Alternative synthesis of compound 5
Figure imgf000156_0002
[00562] To a solution of compound 7 (2 g, 4.31 mmol) and triethylamine (0.902 mL, 6.47 mmol) in DMF (40 mL) was added trityl chloride (1.44 g, 5.18 mmol), and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aq.) and diluted with diethyl ether. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–50% ethyl acetate in hexanes) to obtain compound 8 as a white foam (2.52 g, 83%). Synthesis of compound 8
Figure imgf000157_0001
[00563] To a solution of compound 5 (2.3 g, 3.26 mmol) in THF (30 mL) was added dropwise TBAF (1 M in THF; 3.91 mL, 3.91 mmol) at 0 °C. The mixture was stirred at room temperature for 5 h and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–80% ethyl acetate in hexanes) to obtain compound 8 as a white foam (1.39 g, 91%). [00564] 1H NMR (500 MHz, DMSO-d6) δ 11.17 (d, J = 2.4 Hz, 1H), 7.16–7.50 (m, 16H), 5.40 (dd, J = 2.4 and 8.1 Hz, 1H), 4.78–4.84 (m, 1H), 4.50–4.52 (m, 1H), 3.22–3.26 (m, 1H), 3.16–3.17 (m, 1H), 3.09–3.12 (m, 1H), 2.10–2.17 (m, 1H), 1.76–1.80 (m, 1H), 1.23–1.32 (m, 2H), 0.99 (t, J = 11.3 Hz, 1H).13C NMR (101 MHz, DMSO-d6) δ 162.92, 150.81, 141.87, 128.69, 127.63, 126.04, 100.92, 76.75, 63.70, 52.55, 52.32, 51.14, 38.72, 31.17. HRMS calc. for C29H30N3O3 [M + H]+ 468.2287, found 468.2308. Synthesis of compound 9
Figure imgf000157_0002
[00565] Phosphorus oxychloride (0.632 mL, 6.80 mmol) was added dropwise to a solution of 1,2,4-triazole (3.76 g, 54.4 mmol) and Et3N (7.58 mL, 54.4 mmol) in CH3CN (10 mL) at 0 °C. After stirring for 30 min, a solution of compound 5 (1.6 g, 2.27 mmol) in CH3CN (10 mL) was added dropwise to the reaction mixture at 0 °C. After stirring for 10 min at 0 °C, the reaction was stirred at room temperature for 3 h. The reaction was quenched with saturated NaHCO3 (aq.) and diluted with ethyl acetate. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. [00566] The residue was dissolved in THF (10 mL) and ammonium hydroxide (10 mL), and the mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and water. The organic layer was washed with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–60% ethyl acetate in hexanes) to obtain compound 9 as a white foam (1.0 g, 63%). [00567] 1H NMR (500 MHz, DMSO-d6) δ 7.16–7.54 (m, 26H),6.91–6.95 (m, 2H), 5.51 (d, 7.4 Hz, 1H), 4.90–4.96 (m, 1H), 3.59 (dd, J = 4.6 and 10.1 Hz, 1H), 3.42–3.44 (m, 1H), 3.23 (dd, J = 9.0 and 10.1 Hz, 1H), 3.08–3.10 (m, 1H), 2.28–2.37 (m, 1H), 1.63–1.67 (m, 1H), 1.07–1.21 (m, 2H), 0.97 (t, J = 11.2 Hz, 1H), 0.85 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 165.01, 155.27, 141.98, 134.95, 134.92, 132.99, 132.85, 129.74, 128.62, 127.79, 127.76, 127.60, 125.98, 93.24, 76.75, 66.11, 53.11, 52.03, 51.67, 38.61, 30.94, 26.52, 18.68. HRMS calc. for C45H49N4O2Si [M + H]+ 705.3625, found 705.3621. Synthesis of compound 10
Figure imgf000158_0001
[00568] To a solution of compound 9 (300 mg, 0.426 mmol) in pyridine (4 mL) was added acetic anhydride (60.3 μL, 0.638 mmol), and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aq.) and diluted with ethyl acetate. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 10 as a white foam (302 mg, 95%). [00569] 1H NMR (500 MHz, DMSO-d6) δ 10.71 (s, 1H), 7.86 (d, 7.4 Hz, 1H),7.27–7.53 (m, 25H),6.98 (d, 7.4 Hz, 1H), 4.96–5.02 (m, 1H), 3.60 (dd, J = 4.5 and 10.3 Hz, 1H), 3.45–3.47 (m, 1H), 3.16–3.25 (m, 2H), 2.33–2.40 (m, 1H), 2.04 (s, 3H), 1.74–1.76 (m, 1H), 1.17–1.29 (m, 2H), 0.97 (t, J = 11.2 Hz, 1H), 0.85 (s, 9H). 13C NMR (126 MHz, DMSO-d6) δ 170.80, 161.49, 154.79, 146.46, 134.95, 134.93, 132.95, 132.80, 129.77, 128.66, 127.81, 127.78, 127.65, 126.04, 95.11, 76.80, 66.03, 53.35, 52.97, 51.67, 38.52, 30.76, 26.52, 18.69. HRMS calc. for C47H50N4NaO3Si [M + Na]+ 769.3550, found 769.3527. Synthesis of compound 11
Figure imgf000159_0001
[00570] To a solution of compound 10 (440 mg, 0.623 mmol) in THF (5 mL) at 0 °C, TBAF (1 M in THF; 0.748 mL, 0.748 mmol) was added dropwise. The mixture was stirred at room temperature for 5 h and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–80% ethyl acetate in hexanes) to obtain compound 11 as a white foam (290 mg, quant). [00571] 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 7.91 (d, J =7.4 Hz, 1H), 7.16–7.39 (m, 15H),7.02 (d, 7.4 Hz, 1H), 4.95–5.02 (m, 1H), 4.53–4.55 (m, 1H), 3.12–3.29 (m, 4H), 2.10–2.16 (m, 1H), 2.05 (s, 3H), 1.82–1.85 (m, 1H), 1.21–1.33 (m, 2H), 1.03 (t, J = 11.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 170.83, 161.50, 154.85, 146.54, 128.71, 127.66, 126.06, 95.15, 76.84, 63.72, 53.55, 53.00, 51.29, 38.73, 31.73, 24.26. HRMS calc. for C31H33N4O3 [M + H]+ 509.2553, found 509.2546. Synthesis of compound 12
Figure imgf000159_0002
[00572] To a solution of (1S,4R)-4-amino-2-cyclopentene-1-methanol hydrochloride (1; 1.0 g, 6.68 mmol) and triethyl amine (1.86 mL, 13.4 mmol) in n-butanol (60 mL) was added 5-amino- 4,6-dichloropyrimidine (1.64 g, 10.0 mmol), and the mixture was refluxed for 12 h. After the reaction mixture was concentrated under vacuum, the crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 12 as a white solid (1.35 g, 84%). [00573] 1H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 1H), 6.72 (d, J =7.1 Hz, 1H), 5.90–5.92 (m, 1H), 5.78–5.79 (m, 1H), 5.03–5.08 (m, 3H), 4.62 (t, J =5.3 Hz, 1H), 3.35–3.42 (m, 2H), 2.69–2.75 (m, 1H), 2.39–2.46 (m, 1H), 1.32 (ddd, J = 6.6, 6.6 and 13.2 Hz, 1H).13C NMR (101 MHz, DMSO- d6) δ 151.19, 145.50, 136.70, 135.26, 132.38, 123.37, 65.09, 56.62, 47.33, 34.34. HRMS calc. for C10H14ClN4O [M + H]+ 241.0856, found 241.0845. Synthesis of compound 13
Figure imgf000160_0001
[00574] A mixture of compound 12 (1.35 g, 5.61 mmol), triethylorthoformate (10 mL), and conc. HCl (0.5 mL) was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum, and 0.5 N HCl (aq.) was added. The mixture was stirred at room temperature for 1 h. The mixture was neutralized with 1 N NaOH (aq.) and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 13 as a white solid (1.27 g, 91%). [00575] 1H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.60 (s, 1H),6.18–6.20 (m, 1H), 5.95– 5.97 (m, 1H), 5.70–5.74 (m, 1H), 4.72 (t, J = 5.3 Hz, 1H), 3.42–3.51 (m, 2H), 2.89–2.94 (m, 1H), 2.72 (ddd, J = 8.8, 8.8 and 13.8 Hz, 1H), 1.73 (ddd, J = 5.5, 5.5 and 13.8 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 151.50, 151.29, 148.88, 145.72, 139.27, 131.10, 128.82, 63.64, 60.17, 47.78, 33.85. HRMS calc. for C11H12ClN4O [M + H]+ 251.0700, found 251.0709. Synthesis of compound 14
Figure imgf000160_0002
[00576] To a solution of compound 13 (5 g, 20.0 mmol) and imidazole (4.07 g, 59.8 mmol) in DMF (100 mL) was added tert-butyldiphenylsilyl chloride (7.13 g, 25.9 mmol), and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aq.) and diluted with diethyl ether. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–25% ethyl acetate in hexanes) to obtain compound 14 as a white foam (9.51 g, 98%). [00577] 1H NMR (600 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.50 (s, 1H), 7.36–7.58 (m, 10H), 6.22– 6.24 (m, 1H), 6.04–6.06 (m, 1H), 5.71–5.75 (m, 1H), 3.77 (dd, J = 6.2 and 10.0 Hz, 1H), 3.69 (dd, J = 6.6 and 10.0 Hz, 1H), 3.05–3.11 (m, 1H), 2.74 (ddd, J = 8.6, 8.6 and 13.7 Hz, 1H), 1.78 (ddd, J = 6.2, 6.2 and 13.7 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 151.99, 151.72, 149.49, 145.90, 138.60, 135.48, 133.48, 133.44, 131.76, 130.27, 130.25, 129.89, 128.30, 128.27, 66.78,60.92, 47.97, 34.27, 27.09, 19.27. HRMS calc. for C27H30ClN4OSi [M + H]+ 489.1877, found 489.1888. Synthesis of compound 15
Figure imgf000161_0001
[00578] To a mixture of compound 14 (6 g, 12.3 mmol) and N-methylmorpholine-N-oxide (3.59 g, 30.7 mmol) in a 5:1 acetone-water mixture (120 mL) was added osmium tetroxide (4 wt% in H2O, 3 mol%). The mixture was stirred at room temperature for 24 h, and the reaction quenched with saturated Na2S2O3 (aq.), and stirred for 30 min. The reaction mixture was diluted in ethyl acetate, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were dried (Na2SO4) and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (60–100% ethyl acetate in hexanes) to obtain compound 15 as a white foam (5.54 g, 86%). [00579] 1H NMR (600 MHz, DMSO-d6) δ 8.75–8.76 (m, 1H), 8.67–8.69 (m, 1H), 7.42–7.68 (m, 10H), 5.20 (q, J = 8.3 Hz, 0.5H), 5.09–5.11 (m, 1.5H), 4.82–4.90 (m, 1H), 4.45 (ddd, J = 4.9, 4.9 and 9.4 Hz, 0.5H), 4.24 (ddd, J = 4.0, 4.0 and 8.6 Hz, 0.5H), 4.12 (q, J = 4.0 Hz, 0.5H), 3.99– 4.02 (m, 1H), 3.71–3.80 (m, 1.5H), 2.40–2.45 (m, 0.5H), 2.21–2.33 (m, 1.5H), 1.99–2.05 (m, 0.5H), 1.86–1.92 (m, 0.5H), 1.03 (s, 4.5H), 1.00 (s, 4.5H).13C NMR (151 MHz, DMSO-d6) δ 152.96, 152.52, 151.62, 151.46, 149.55, 149.03, 148.69, 147.47, 135.58, 135.56, 135.54, 133.82, 133.79, 133.56, 132.00, 130.89, 130.32, 130.24, 130.22, 128.36, 128.33, 128.32, 74.82, 72.49, 71.79, 71.61, 65.53, 64.07, 61.00, 54.19, 45.71, 42.23, 34.01, 28.74, 27.15, 27.12, 19.37, 19.31. HRMS calc. for C27H32ClN4O3Si [M + H]+ 523.1932, found 523.1918. Synthesis of compound 16
Figure imgf000162_0001
[00580] To a vigorously stirred suspension of chromatographic grade silica gel (20.0 eq.) in MeOH (50 mL) was added dropwise a solution of sodium periodate (2.25 g, 10.5 mmol) in water (5 mL) followed by dropwise addition of a solution of compound 15 (6 g, 12.5 mmol) in MeOH (50 mL). The resulting mixture was stirred at room temperature for 3 h and filtered through a pad of celite. To the filtrate were added benzylamine (1.15 mL, 10.5 mmol), sodium cyanoborohydride (1.20 g, 19.1 mmol), molecular sieves (4 Å, 300 mg/mmol). Subsequently, acetic acid (1.09 mL, 12.5 mmol) was added dropwise. The mixture was stirred at room temperature for 12 h and filtered. The filtrate was concentrated under vacuum. The residue was partitioned between saturated NaHCO3 (aq.) and EtOAc, and the organic layer was washed with water and with brine, dried over Na2SO4, and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–70% ethyl acetate in hexanes) to obtain compound 16 as a white foam (3.82 g, 67%). [00581] 1H NMR (600 MHz, DMSO-d6) δ 8.76 (s, 1H), 8.75 (s, 1H), 7.53–7.55 (m, 4H),7.38– 7.45 (m, 6H), 7.30–7.31 (m, 4H), 7.22–7.24 (m, 1H), 4.73–4.78 (m, 1H), 3.58–3.66 (m, 2H),3.46– 3.50 (m, 2H),3.06–3.15 (m, 2H), 2.49–2.53 (m, 1H), 2.02–2.08 (m, 2H), 1.87 (q, J = 12.2 Hz, 1H), 1.75 (t, J = 11.0 Hz, 1H), 0.90 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 152.04, 151.69, 149.61, 146.51, 138.15, 135.53, 135.48, 133.40, 133.31, 131.54, 130.32, 130.30, 129.34, 128.62, 128.34, 127.47, 66.55, 62.21, 57.72, 55.57, 52.98, 38.46, 32.27, 27.05, 19.22. HRMS calc. for C34H39ClN5OSi [M + H]+ 596.2612, found 596.2606. Synthesis of compound 17
Figure imgf000163_0001
[00582] A mixture of compound 16 (500 mg, 0.839 mmol), ammonium hydroxide solution (5 mL), and 1,4-dioxane (5 mL) was stirred and heated in a microwave reactor at 95 ºC for 3 h. The mixture was concentrated under vacuum, and the crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 17 as a white foam (414 mg, 86%). [00583] 1H NMR (600 MHz, DMSO-d6) δ 8.18 (s, 1H), 8.13 (s, 1H), 7.54–7.56 (m, 4H),7.39– 7.46 (m, 6H), 7.31–7.31 (m, 4H), 7.22–7.25 (m, 3H), 4.57–4.62 (m, 1H), 3.59–3.66 (m, 2H),3.46– 3.49 (m, 2H), 3.06–3.08 (m, 2H), 2.44 (t,J = 10.7 Hz 1H), 1.94–2.05 (m, 2H), 1.80 (q, J = 12.2 Hz, 1H), 1.75 (t, J = 11.0 Hz, 1H), 0.92 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 156.53, 152.70, 149.64, 139.63, 138.30, 135.53, 135.48, 133.49, 133.40, 130.31, 130.29, 129.34, 128.62, 128.34, 127.44, 119.43, 66.66, 62.26, 58.27, 55.69, 51.88, 38.59, 32.62, 27.08, 19.24. HRMS calc. for C34H41N6OSi [M + H]+ 577.3111, found 577.3122. Synthesis of compound 18
Figure imgf000163_0002
[00584] To a solution of compound 17 (2.34 g, 4.06 mmol) in pyridine (40 mL) was added dropwise benzoyl chloride (0.518 mL, 4.46 mmol), and the mixture was stirred at 0 °C for 3 h. The reaction was quenched with dry MeOH and concentrated under vacuum. The residue was dissolved in ethyl acetate and washed with saturated NaHCO3 (aq.), water, and brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 18 (N-Bz) as a white foam (1.63 g, 59%) and compound 18A (N-Bz2) as a white foam (986 mg, 31.0%). [00585] Compound 18 (N-Bz): 1H NMR (600 MHz, DMSO-d6) δ 11.15 (s, 1H), 8.72 (s, 1H), 8.54 (s, 1H), 8.04–8.06 (m, 2H),7.61–7.64 (m, 1H), 7.52–7.57 (m, 6H), 7.39–7.47 (m, 6H), 7.31– 7.33 (m, 4H), 7.23–7.26 (m, 1H), 4.73–4.78 (m, 1H), 3.61–3.69 (m, 2H),3.48–3.51 (m, 2H), 3.08– 3.16 (m, 2H), 2.50–2.53 (m, 1H), 2.01–2.10 (m, 2H), 1.87 (q, J = 12.1 Hz, 1H), 1.76 (t, J = 11.1 Hz, 1H), 0.93 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 166.03, 152.54, 151.68, 150.73, 143.54, 138.21, 135.54, 135.49, 133.91, 133.48, 133.39, 132.84, 130.32, 130.30, 129.37, 128.93, 128.89, 128.63, 128.34, 127.47, 126.06, 66.64, 62.25, 58.04, 55.61, 52.35, 38.56, 32.42, 27.09, 19.25. HRMS calc. for C41H45N6O2Si [M + H]+ 681.3373, found 681.3379. [00586] Compound 18A (N-Bz2): 1H NMR (600 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.66 (s, 1H), 7.77–7.79 (m, 4H),7.54–7.59 (m, 6H), 7.38–7.46 (m, 10H),7.32–7.33 (m, 4H), 7.24–7.27 (m, 1H), 4.72–4.77 (m, 1H), 3.68 (d, J = 13.3 Hz, 1H), 3.61 (dd, J = 5.0 and 10.1 Hz, 1H),3.47–3.50 (m, 2H), 3.16–3.18 (m, 1H), 3.07–3.09 (m, 1H), 2.49–2.53 (m, 1H), 1.99–2.07 (m, 2H), 1.83 (q, J = 12.1 Hz, 1H), 1.75 (t, J = 11.0 Hz, 1H), 0.93 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 172.55, 153.14, 151.79, 151.23, 146.12, 138.20, 135.55, 135.49, 134.02, 133.78, 133.47, 133.37, 130.33, 130.31, 129.48, 129.45, 129.35, 128.65, 128.34, 127.48, 127.40, 66.58, 62.18, 57.81, 55.53, 52.87, 38.49, 32.32, 27.10, 19.25. HRMS calc. for C48H48N6NaO3Si [M + Na]+ 807.3455, found 807.3425. Synthesis of compound 19
Figure imgf000164_0001
[00587] Synthesis of compound 19 was performed under a number of conditions (Table S1) in order to optimize yields. Table 2. Hydrogenation conditions for synthesis of 19
Figure imgf000164_0002
Figure imgf000165_0001
[00588] Entry 1: To a vigorously stirred suspension of 10% palladium on carbon (30 mg), ammonium formate (278 mg, 4.41 mmol), and acetic acid (25.2 μL, 0.441 mmol) in EtOH (5 mL) was added compound 18 (300 mg, 0.441 mmol). The mixture was refluxed for 6 h, and filtered through a pad of celite. The filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–20% MeOH in CH2Cl2) to obtain compound 19 (N-Bz) as a white solid (113 mg, 43.3%) and compound 19A (-NH2) as a yellow solid (62.0 mg, 29%). [00589] Entry 2: To a vigorously stirred suspension of 10% palladium on carbon (30 mg), cyclohexene (0.446 mL, 4.41 mmol), and acetic acid (25.2 μL, 0.441 mmol) in EtOH (5 mL) was added compound 18 (300 mg, 0.441 mmol). The mixture was stirred at 90 °C for 6 h and filtered through a pad of celite. The filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–20% MeOH in CH2Cl2) to obtain compound 19 (N-Bz) as a white solid (131 mg, 50%) and compound 19A (-NH2) as a yellow solid (47.5 mg, 22%). Unreacted compound 18 was recovered as a white foam (27.3 mg, 9.1%). [00590] Entry 3: To a vigorously stirred suspension of 10% palladium on carbon (30 mg) in EtOH (5 mL) was added compound 18 (300 mg, 0.441 mmol) and acetic acid (25.2 μmol, 0.441 mmol ). The mixture was stirred under H2 atmosphere at room temperature for 4 days and filtered through a pad of celite. The filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–10% MeOH in CH2Cl2) to obtain compound 19 (N-Bz) as a white solid (140 mg, 54%) and to recover compound 18 as a white foam (107.3 mg, 36%). [00591] Entry 4: To a vigorously stirred suspension of 10% palladium on carbon (100 mg) and formic acid (0.526 mL, 14.0 mmol) in EtOH (5 mL) was added compound 18 (950 mg, 1.40 mmol). The mixture was refluxed for 6 h and filtered through a pad of celite. The filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–20% MeOH in CH2Cl2) to obtain compound 19 (N-Bz) as a white solid (454 mg, 55%) and compound 19A (-NH2) as a yellow solid (67.6 mg, 10%). [00592] Entry 5: To a vigorously stirred suspension of 10% palladium on carbon (30 mg), cyclohexene (0.446 mL, 4.41 mmol), and acetic acid (25.2 μL, 0.441 mmol) in THF (5 mL) was added compound 18 (300 mg, 0.441 mmol). The mixture was refluxed for 12 h, which resulted in a complex mixture on TLC. [00593] Compound 19 (N-Bz): 1H NMR (600 MHz, DMSO-d6) δ 11.11 (brs, 1H), 8.73 (s, 1H), 8.54 (s, 1H), 8.04–8.06 (m, 2H),7.61–7.65 (m, 5H), 7.53–7.56 (m, 2H), 7.42–7.48 (m, 6H), 4.56– 4.61 (m, 1H), 3.52–3.60 (m, 2H),3.35–3.35 (m, 1H), 3.14–3.21 (m, 2H), 2.91 (t, J = 11.4 Hz, 1H), 2.29 (dd, J = 10.8 and 12.4 Hz, 1H), 2.10–2.13 (m, 1H), 1.96–2.03 (m, 1H), 1.87 (q, J = 11.9 Hz, 1H), 1.01 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 166.17, 152.57, 151.55, 150.68, 143.36, 135.54, 135.52, 133.96, 133.51, 133.50, 132.84, 130.33, 128.91, 128.38, 126.06, 66.90, 53.13, 51.22, 49.02, 33.52, 27.15, 19.33. HRMS calc. for C34H39N6O2Si [M + H]+ 591.2904, found 591.2899. [00594] Compound 19A (-NH2): 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 8.13 (s, 1H), 7.60–7.62 (m, 4H),7.42–7.48 (m, 6H), 7.19 (brs, 2H), 4.41–4.46 (m, 1H), 3.50–3.58 (m, 2H),3.32– 3.33 (m, 1H), 3.13–3.15 (m, 2H), 2.85 (t, J = 11.5 Hz, 1H), 2.29 (dd, J = 10.9 and 12.3 Hz, 1H), 2.04–2.06 (m, 1H), 1.92–1.99 (m, 1H), 1.87 (q, J = 12.1 Hz, 1H), 1.00 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 156.51, 152.64, 149.68, 139.40, 135.51, 133.50, 130.31, 128.36, 119.40, 66.87, 52.55, 51.23, 48.92, 33.63, 27.13, 19.31. HRMS calc. for C27H35N6OSi [M + H]+ 487.2642, found 487.2637. Synthesis of compound 20
Figure imgf000166_0001
[00595] To a solution of compound 19 (350 mg, 0.592 mmol) and triethylamine (0.124 mL, 0.889 mmol) in DMF (6 mL) was added trityl chloride (198 mg, 0.711 mmol), and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aq.) and diluted with diethyl ether. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–50% ethyl acetate in hexanes) to obtain compound 20 as a white foam (482 mg, 98%). [00596] 1H NMR (600 MHz, DMSO-d6) δ 11.18 (s, 1H), 8.74 (s, 1H), 8.43 (s, 1H), 8.01–8.03 (m, 2H),7.18–7.63 (m, 28H), 5.14–5.20 (m, 1H), 3.68 (dd, J = 4.6 and 10.1 Hz, 1H), 3.55–3.57 (m, 1H),3.38–3.39 (m, 1H),3.29 (t, J = 9.5 Hz, 1H), 2.52–2.55 (m, 1H), 2.05–2.08 (m, 1H), 1.69 (q, J = 12.3 Hz, 1H), 1.60 (t, J = 10.7 Hz, 1H),1.10 (t, J = 11.1 Hz, 1H),0.87 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 166.00, 152.52, 151.78, 150.67, 143.05, 135.48, 135.47, 133.77, 133.44, 133.24, 132.88, 130.31, 128.92, 128.91, 128.35, 128.32, 128.21, 126.61, 125.96, 77.24, 66.50, 54.35, 52.44, 52.30, 38.92, 31.80, 27.02, 19.23. HRMS calc. for C53H53N6O2Si [M + H]+ 833.3999, found 833.3967. Synthesis of compound 21
Figure imgf000167_0001
[00597] To a solution of compound 20 (2.8 g, 3.36 mmol) in THF (30 mL) at 0 °C was added dropwise TBAF (1 M in THF; 4.03 mL, 4.03 mmol). The mixture was stirred at room temperature overnight and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (100% ethyl acetate) to obtain compound 21 as a white foam (1.87 g, 94%). [00598] 1H NMR (600 MHz, DMSO-d6) δ 11.13 (s, 1H), 8.74 (s, 1H), 8.48 (s, 1H), 8.02–8.04 (m, 2H),7.18–7.64 (m, 18H), 5.13–5.18 (m, 1H), 4.62 (t, J = 5.3 Hz, 1H), 3.37–3.39 (m, 1H),3.29– 3.35 (m, 2H),3.18–3.22 (m, 1H),2.27–2.34 (m, 1H), 2.16–2.18 (m, 1H), 1.75 (q, J = 12.2 Hz, 1H), 1.66 (t, J = 10.7 Hz, 1H),1.16 (t, J = 11.0 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 166.02, 152.55, 151.75, 150.67, 143.15, 133.86, 132.85, 129.17, 128.91, 128.18, 126.62, 126.00, 77.30, 64.21, 55.39, 54.30, 52.80, 51.87, 39.13, 32.82. HRMS calc. for C37H35N6O2 [M + H]+ 595.2821, found 595.2820. Synthesis of compound 225
Figure imgf000167_0002
[00599] Compound 22 was synthesized essentially as described.5 To a solution of (1S,4R)-4- amino-2-cyclopentene-1-methanol (1; 500 mg, 3.34 mmol) and DIPEA (1.75 mL, 10.0 mmol) in EtOH/H2O (1:1) (16 mL) was added 2-amino-4,6-dichloro-5-formamidopyrimidine (830 mg, 4.01 mmol). The reaction mixture was stirred and heated in a microwave reactor at 140 ºC for 1 h. The reaction was cooled to room temperature, and the solution was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 22 as a white foam (886 mg, quant). [00600] 1H NMR (400 MHz, DMSO-d6) δ 8.02 (s, 1H), 6.89 (s,2H), 6.12–6.14 (m, 1H), 5.88– 5.91 (m, 1H), 5.42–5.46 (m, 1H), 4.70–4.73 (m, 1H), 3.43–3.46 (m, 2H), 2.83–2.90 (m, 1H), 2.62 (ddd, J = 8.8, 8.8 and 13.8 Hz, 1H), 1.63 (ddd, J = 5.5, 5.5 and 13.8 Hz, 1H).13C NMR (126 MHz, DMSO-d6) δ 159.66, 153.67, 149.28, 141.11, 138.86, 129.20, 123.56, 63.76, 59.02, 47.71, 33.88. HRMS calc. for C11H13ClN5O [M + H]+ 266.0809, found 266.0805. Synthesis of compound 23
Figure imgf000168_0001
[00601] To a solution of compound 22 (1.77 g, 6.68 mmol) and imidazole (1.36 g, 20.0 mmol) in DMF (50 mL) was added tert-butyldiphenylsilyl chloride (2.02 g, 7.35 mmol), and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aq.) and diluted with diethyl ether. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–50% ethyl acetate in hexanes) to obtain compound 23 as a white solid (3.37 g, 83%). [00602] 1H NMR (600 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.56–7.59 (m, 4H),7.38–7.47 (m, 6H), 6.89 (s,2H), 6.19–6.20 (m, 1H), 5.99–6.01 (m, 1H), 5.44–5.47 (m, 1H), 3.71 (dd, J = 6.2 and 10.0 Hz, 1H), 3.66 (dd, J = 6.5 and 10.0 Hz, 1H), 3.01–3.05 (m, 1H), 2.65 (ddd, J = 8.5, 8.5 and 13.7 Hz, 1H), 1.63 (ddd, J = 6.1, 6.1 and 13.7 Hz, 1H), 0.98 (s, 9H).13C NMR (151 MHz, DMSO-d6) δ 160.17, 154.16, 149.85, 140.96, 138.47, 135.49, 133.49, 133.44, 130.31, 130.30, 130.27, 128.35, 128.34, 124.12, 66.82, 59.50, 47.82, 34.44, 27.12, 19.29. HRMS calc. for C27H31ClN5OSi [M + H]+ 504.1986, found 504.1970. Synthesis of compound 24
Figure imgf000169_0001
[00603] 3-Hydroxypropionitrile (3.39 mL, 49.6 mmol) was dissolved in THF (50 mL) and cooled to 0 °C. Sodium hydride (60% in mineral oil; 1.90 g, 49.6 mmol) was added in portions, and the mixture was stirred at room temperature for 30 min and then cooled to 0 °C. A solution of compound 23 (5.56 g, 11.0 mmol) in THF (50 mL) was added dropwise at 0 °C, and the mixture was stirred at room temperature. After 12 h, the reaction was quenched by addition of saturated NH4Cl (aq.). The 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 CH2Cl2) to obtain compound 24 as a brown solid (5.28 g, 99%). [00604] 1H NMR (600 MHz, DMSO-d6) δ 10.6 (s, 1H), 7.40–7.60 (m, 11H), 6.46 (s,2H), 6.14– 6.15 (m, 1H), 5.93–5.94 (m, 1H), 5.34–5.36 (m, 1H), 3.67–3.68 (m, 2H), 2.97–3.02 (m, 1H), 2.60 (ddd, J = 8.3, 8.3 and 13.6 Hz, 1H), 1.60 (ddd, J = 6.2, 6.2 and 13.6 Hz, 1H), 0.98 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 157.36, 153.98, 151.29, 138.04, 135.50, 134.94, 133.49, 133.44, 130.72, 130.31, 128.36, 117.26, 66.80, 58.94, 47.73, 34.87, 27.12, 19.30. HRMS calc. for C27H32ClN5O2Si [M + H]+ 486.2325, found 486.2313. Synthesis of compound 25
Figure imgf000169_0002
[00605] To a suspension of compound 24 (5 g, 10.3 mmol) in pyridine (50 mL) at 0 °C was added dropwise isobutyryl chloride (1.61 mL, 15.4 mmol), and the mixture was stirred at room temperature overnight. The reaction was quenched with dry MeOH and concentrated under vacuum. The residue was dissolved in ethyl acetate and washed with saturated NaHCO3 (aq.), water, and brine and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 25 as a brown foam (4.16 g, 73%). [00606] 1H NMR (600 MHz, DMSO-d6) δ 12.08 (s, 1H), 11.67 (s, 1H), 7.73 (s, 1H), 7.57–7.59 (m, 4H),7.39–7.41 (m, 6H),6.20–6.21 (m, 1H), 6.00–6.01 (m, 1H), 5.43–5.46 (m, 1H), 3.66–3.71 (m, 2H), 3.02–3.06 (m, 1H), 2.79 (sep, J = 6.8 Hz, 1H), 2.66 (ddd, J = 8.5, 8.5 and 13.7 Hz, 1H), 1.66 (ddd, J = 6.1, 6.1 and 13.7 Hz, 1H), 1.11–1.13 (m, 6H),0.98 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 180.61, 155.41, 148.73, 148.29, 138.61, 137.37, 135.49, 133.46, 133.42, 130.33, 130.32, 130.25, 128.35, 120.80, 66.78, 59.62, 47.83, 35.16, 34.88, 27.12, 19.35, 19.34, 19.30. HRMS calc. for C31H38N5O3Si [M + H]+ 556.2744, found 556.2741. Synthesis of compound 26
Figure imgf000170_0001
[00607] To a mixture of compound 25 (4 g, 7.20 mmol) and N-methylmorpholine-N-oxide (2.11 g, 18.0 mmol) in a 5:1 acetone-water mixture (60 mL) was added osmium tetroxide (4 wt% in H2O, 3 mol%). The mixture was stirred at room temperature for 24 h, and the reaction was quenched with a saturated Na2S2O3 (aq.), and stirred for 30 min. The reaction mixture was diluted in ethyl acetate, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–10% MeOH in CH2Cl2) to obtain compound 26 as a brown foam (3.91 g, 92%). [00608] 1H NMR (600 MHz, DMSO-d6) δ 12.01–12.04 (m, 1H), 11.65 (s, 0.2H), 11.62 (s, 0.8H), 8.08 (s, 0.8H), 7.98 (s, 0.2H), 7.62–7.67 (m, 4H), 7.42–7.48 (m, 6H), 5.10 (d, J = 6.1 Hz, 0.8H), 5.01 (d, J = 5.4 Hz, 0.2H),4.96 (d, J = 4.0 Hz, 0.2H),4.80–4.84 (m, 0.2H), 4.68–4.72 (m, 1.6H),4.26 (ddd, J = 5.6, 5.6 and 9.1 Hz, 0.8H), 4.08–4.13 (m, 0.4H),3.93–3.99 (m, 1H),3.69–3.78 (m, 1.8H),2.77 (sep, J = 6.9 Hz, 1H), 2.26–2.34 (m, 1.2H), 2.14–2.19 (m, 0.8H), 1.81–1.87 (m, 0.2H), 1.52–1.57 (m, 0.8H), 1.12–1.13 (m, 6H), 1.01–1.02 (m, 9H).13C NMR (151 MHz, DMSO- d6) δ 180.51, 180.48, 155.44, 155.39, 149.59, 149.49, 148.13, 148.01, 140.59, 138.04, 135.55, 135.52, 133.82, 133.78, 133.58, 130.34, 130.26, 130.24, 128.39, 128.37, 128.34, 128.32, 120.56, 119.71, 75.36, 72.59, 71.82, 71.81, 65.82, 64.23, 58.60, 53.58, 45.53, 41.99, 35.19, 35.18, 34.00, 30.54, 27.16, 19.36, 19.34, 19.31. HRMS calc. for C31H40N5O5Si [M + H]+ 590.2799, found 590.2780. Synthesis of compound 27
Figure imgf000171_0001
[00609] To a vigorously stirred suspension of chromatographic grade silica gel (20.0 eq.) in MeOH (10 mL) was added dropwise a solution of sodium periodate (399 mg, 1.87 mmol) in water (5 mL), followed by a solution of compound 26 (1 g, 1.70 mmol) in MeOH (10 mL). The resulting mixture was stirred at room temperature for 3 h and filtered through a pad of celite. To the filtrate was added benzylamine (0.204 mL, 1.87 mmol), sodium cyanoborohydride (213 mg, 3.39 mmol), molecular sieves (4 Å, 300 mg/mmol. Acetic acid (0.194 mL, 3.39 mmol) was added dropwise. The mixture was stirred at room temperature for 12 h and filtered. The filtrate was concentrated under vacuum. The residue was partitioned between saturated NaHCO3 (aq.) and EtOAc, and the organic layer was washed with water and with brine, and dried over Na2SO4, and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (50–100% ethyl acetate in hexanes) to obtain compound 27 as a white foam (788 mg, 70%). [00610] 1H NMR (600 MHz, DMSO-d6) δ 12.06 (s, 1H), 11.64 (s, 1H), 8.07 (s, 1H), 7.40–7.55 (m, 10H),7.24–7.35 (m, 5H), 4.45–4.50 (m, 1H), 3.59–3.67 (m, 2H),3.45–3.48 (m, 2H), 3.06–3.07 (m, 2H), 2.78 (sep, J = 6.9 Hz, 1H),2.31 (t, J = 10.9 Hz, 1H), 1.96–1.98 (m, 2H), 1.62–1.70 (m, 2H),1.11–1.12 (m, 6H), 0.92 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 180.60, 155.36, 148.63, 148.24, 138.17, 138.08, 135.53, 135.48, 133.44, 133.31, 130.36, 130.33, 129.30, 128.68, 128.36, 128.35, 127.50, 120.45, 66.61, 62.20, 58.74, 55.52, 51.51, 38.65, 35.16, 32.77, 27.08, 19.37, 19.31, 19.23. HRMS calc. for C38H47N6O3Si [M + H]+ 663.3479, found 663.3494. Synthesis of compound 28
Figure imgf000171_0002
[00611] To a vigorously stirred suspension of 10% palladium on carbon (70 mg) and formic acid (0.398 mL, 10.6 mmol) in EtOH (10 mL) was added compound 27 (700 mg, 1.06 mmol). The mixture was refluxed for 6 h and filtered through a pad of celite. The filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–10% MeOH in CH2Cl2) to obtain compound 28 as a white foam (318 mg, 53%). [00612] To a vigorously stirred suspension of 10% palladium on carbon (300 mg), 1,3- cyclohexadiene (4.31 mL, 45.3 mmol), and acetic acid (0.259 mL, 4.53 mmol) in EtOH (45 mL) was added compound 27 (3.0 g, 45.3 mmol). The mixture was refluxed overnight and filtered through a pad of celite. The filtrate was concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–10% MeOH in CH2Cl2) to obtain compound 28 as a white foam (1.46 g, 56%). [00613] 1H NMR (600 MHz, DMSO-d6) δ 11.63 (brs, 1H), 8.05 (s, 1H), 7.59–7.61 (m, 4H), 7.42–7.47 (m, 6H),4.26–4.31 (m, 1H), 3.49–3.56 (m, 2H),3.36–3.36 (m, 1H), 3.11–3.16 (m, 2H), 2.73–2.81 (m, 2H), 2.24 (dd, J = 11.0 and 12.2 Hz, 1H),2.04–2.06 (m, 1H), 1.86–1.93 (m, 1H), 1.67 (q, J = 12.2 Hz, 1H), 1.12 (d, J = 6.8 Hz, 6H),0.99 (s, 9H).13C NMR (151 MHz, DMSO-d6) δ 180.57, 155.41, 148.71, 148.14, 137.95, 135.52, 135.51, 133.47, 133.43, 130.35, 130.33, 128.38, 120.51, 66.86, 52.51, 51.53, 48.97, 35.16, 33.95, 27.14, 19.36, 19.33, 19.30. HRMS calc. for C31H41N6O3Si [M + H]+ 573.3009, found 573.3023. Synthesis of compound 29
Figure imgf000172_0001
[00614] To a solution of compound 28 (218 mg, 0.381 mmol) and triethylamine (79.6 μL, 0.571 mmol) in DMF (4 mL) was added trityl chloride (127 mg, 0.457 mmol), and the mixture was stirred at room temperature for 3 h. The reaction mixture was quenched with saturated NaHCO3 (aq.) and diluted with diethyl ether. The organic layer was washed with water and with brine, dried (Na2SO4), and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–60% ethyl acetate in hexanes) to obtain compound 29 as a white foam (263 mg, 85%). [00615] 1H NMR (600 MHz, DMSO-d6) δ 12.02 (s, 1H),11.59 (s, 1H), 7.92 (s, 1H), 7.18–7.54 (m, 25H),4.83–4.89 (m, 1H), 3.67 (dd, J = 4.4 and 10.3 Hz, 1H), 3.51–3.53 (m, 1H), 3.25–3.29 (m, 2H),2.88 (sep, J = 6.8 Hz, 1H), 2.35–2.42 (m, 1H), 2.00–2.02 (m, 1H), 1.50–1.56 (m, 2H),1.15– 1.19(m, 6H),1.07 (t, J = 11.2 Hz, 1H),0.86 (s, 9H). 13C NMR (151 MHz, DMSO-d6) δ 180.52, 155.31, 148.60, 148.24, 137.85, 135.45, 135.44, 133.45, 133.21, 130.31, 129.18, 128.33, 128.32, 126.62, 120.40, 77.23, 66.44, 54.66, 52.31, 52.06, 39.11, 35.21, 32.15, 27.02, 19.58, 19.20, 19.14. HRMS calc. for C50H55N6O3Si [M + H]+ 815.4105, found 815.4120. Synthesis of compound 30
Figure imgf000173_0001
[00616] To a solution of compound 29 (2.9 g, 3.56 mmol) in THF (35 mL) at 0 °C was added dropwise TBAF (1 M in THF; 4.27 mL, 4.27 mmol). The mixture was stirred at room temperature for 24 h and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (0–10% MeOH in CH2Cl2) to obtain compound 30 as a white foam (1.90 g, 93%). [00617] 1H NMR (600 MHz, DMSO-d6) δ 12.03 (s, 1H),11.65 (s, 1H), 7.97 (s, 1H), 7.16–7.32 (m, 15H),4.83–4.88 (m, 1H), 4.63 (d, J = 5.2 Hz, 1H), 3.24–3.32 (m, 3H), 3.14–3.18 (m, 1H),2.87 (sep, J = 6.8 Hz, 1H), 2.15–2.22 (m, 1H), 2.08–2.10 (m, 1H), 1.52–1.60 (m, 2H), 1.08–1.18 (m, 7H). 13C NMR (151 MHz, DMSO-d6) δ 180.54, 155.34, 148.62, 148.24, 137.92, 128.25, 126.63, 120.37, 77.24, 64.09, 54.49, 52.29, 51.79, 39.30, 35.22, 33.31, 19.57, 19.16. HRMS calc. for C34H37N6O3 [M + H]+ 577.2927, found 577.2918. Synthesis of compound 8a
Figure imgf000174_0001
[00618] To a solution of compound 8 (230 mg, 0.49 mmol) in THF (5 mL) at ice-salt bath temperature was added dropwise LDA (2 M in THF; 0.61 mL, 1.23 mmol). The mixture was stirred at ice-salt bath temperature for 30 min, and then the reaction was quenched with 0.5 mL saturated aq. NH4Cl solution. Then the reaction mixture was diluted with 75 mL chilled DCM and then washed with 15 mL saturated aq NH4Cl solution and 20 mL cold water. The organic layer was dried over Na2SO4 and concentrated under vacuum. The crude residue was purified by column chromatography on silica gel (2 % MeOH, 10 % acetone in DCM) to obtain compound 8a as a white foam (148 mg, 51%) and compound 8b as minor side product (32 mg, 9%). [00619] Compound 8a: 1H NMR (300 MHz, CDCl3) δ 9.01 (s, 1H), 7.52 – 7.37 (m, 6H), 7.29 (d, J = 7.4 Hz, 5H), 7.17 (t, J = 7.3 Hz, 3H), 6.96 (dd, J = 8.1, 2.3 Hz, 1H), 5.61 (ddd, J = 8.0, 2.3, 1.1 Hz, 1H), 5.10 – 5.00 (m, 1H), 4.00 – 3.88 (m, 2H), 3.40 – 3.26 (m, 2H), 2.62 (dd, J = 13.8, 3.5 Hz, 6H), 2.56-2.51 (m, 1H), 2.14 – 2.02 (m, 1H), 1.36 (t, J = 10.8 Hz, 2H), 1.17 – 1.12 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 170.90, 161.96, 155.62, 144.68, 129.14, 128.86, 128.69, 128.06, 128.02, 127.94, 127.86, 127.34, 126.51, 96.87, 69.26, 53.45, 51.19, 51.02, 36.97, 36.84, 36.74, 36.70, 36.59, 32.74, 29.41, 25.0. 31P NMR (121 MHz, CDCl3) δ 18.06, 17.99. HRMS calc. for C31H34N4O4PClNa [M + Na]+ 615.1904, found 615.1905. [00620] Compound 8b: 1H NMR (300 MHz, CD2Cl2) δ 7.54 – 7.38 (m, 7H), 7.28 (t, J = 7.8 Hz, 7H), 7.17 (t, J = 7.3 Hz, 3H), 6.01 (d, J = 7.2 Hz, 1H), 5.23 – 5.10 (m, 1H), 4.04 – 3.83 (m, 2H), 3.46 – 3.27 (m, 2H), 2.77 (dd, J = 13.8, 0.8 Hz, 6H), 2.57 (dd, J = 13.9, 3.0 Hz, 8H), 2.16 – 2.04 (m, 1H), 1.33 (dtd, J = 8.2, 5.3, 4.7, 2.6 Hz, 1H), 1.25 – 1.15 (m, 2H).13C NMR (75 MHz, CD2Cl2) δ 165.76, 165.66, 154.89, 154.86, 147.12, 129.42, 128.15, 126.71, 95.60, 95.50, 77.79, 69.52, 51.42, 51.20, 37.12, 37.03, 36.94, 36.87, 36.82, 36.79, 36.76, 36.74, 32.50, 32.45. 31P NMR (121 MHz, CD2Cl2) δ 17.67, 17.63, 10.47, 10.43. HRMS calc. for C33H39N5O5P2Cl2Na [M + Na]+ 740.1701, found 740.1703. Compound 11a
Figure imgf000175_0001
[00621] Compound 11a was synthesized from 230 mg of compound 11 to yield 11a (140 mg, 49 %) following the same synthesis procedure as that of 8a. [00622] 1H NMR (300 MHz, CDCl3) δ 10.07 (s, 1H), 7.51 – 7.38 (m, 5H), 7.38 – 7.26 (m, 5H), 7.24 – 7.09 (m, 5H), 5.23 (t, J = 11.7 Hz, 1H), 4.00 - 3.94 (m, 2H), 3.40-3.30 (m, 2H), 2.60 (dd, J = 13.8, 4.6 Hz, 6H), 2.23 (s, 3H), 2.13 (m, 1H), 1.35 (t, J = 10.7 Hz, 1H), 1.22 – 1.05 (m, 2H).13C NMR (75 MHz, CDCl3) δ 170.90, 161.98, 155.51, 144.71, 144.10, 129.12, 128.86, 128.06, 128.04, 127.95, 127.88, 127.03, 126.51, 96.85, 69.4, 53.43, 51.19, 51.01, 36.75, 36.71, 32.76, 25.03. 31P NMR (121 MHz, CDCl3) δ 18.03, 17.93. HRMS calc. for C33H37N5O4PClNa [M + Na]+ 656.2169, found 656.2166. Compound 21a
Figure imgf000175_0002
[00623] Compound 21a was synthesized from 230 mg of 21 to yield 21a (131 mg, 47 %) following the same as synthesis procedure as that of 8a. [00624] 1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 8.77 (s, 1H), 8.00 (d, J = 7.2 Hz, 2H), 7.85 (s, 1H), 7.61 – 7.38 (m, 9H), 7.34 – 7.23 (m, 7H), 7.17 (t, J = 7.2 Hz, 3H), 5.18 (t, J = 11.7 Hz, 1H), 4.08 - 3.91 (m, 2H), 3.60 – 3.50 (m, 1H), 3.40 (d, J = 11.5 Hz, 1H), 2.62 (dd, J = 13.8, 5.6 Hz, 6H), 2.39 (d, J = 11.8 Hz, 1H), 1.72 (t, J = 10.9, 1H), 1.55 (dd, J = 12.3, 3.9 Hz, 1H), 1.37 – 1.25 (m, 1H).13C NMR (75 MHz, CDCl3) δ 164.82, 152.55, 152.05, 149.60, 140.55, 133.76, 132.85, 129.10, 128.94, 128.02, 127.97, 126.58, 123.24, 69.27, 69.20, 54.13, 52.30, 51.13, 50.92, 36.97, 36.91, 36.77, 36.74, 36.71, 33.53, 33.46.31P NMR (121 MHz, CDCl3) δ 18.03, 17.97. HRMS calc. for C39H40N7O3PCl [M + H]+ 720.2619 found 720.2618. Compound 30a
Figure imgf000176_0001
[00625] Compound 30a was synthesized from 230 mg of 30 to yield 30a (98 mg, 35 %) following the same synthesis procedure as that of 8a. [00626] 1H NMR (300 MHz, CDCl3) δ 12.01 (d, J = 3.1 Hz, 1H), 8.85 (d, J = 10.8 Hz, 1H), 7.50 – 7.38 (m, 6H), 7.31 -7.25 (m, 5H), 7.17 (t, J = 7.3 Hz, 3H), 4.75 (t, J = 11.8 Hz, 1H), 4.04 – 3.85 (m, 2H), 3.59 – 3.50 (m, 1H), 3.38 (d, J = 11.0 Hz, 1H), 2.79 – 2.69 (m, 1H), 2.63 (dd, J = 13.9, 2.8 Hz, 6H), 2.30 – 2.15 (m, 2H), 1.54 – 1.42 (m, 1H), 1.35 – 1.23 (m, 7H). 13C NMR (75 MHz, CDCl3) δ 178.55, 178.50, 155.79, 148.26, 147.35, 136.20, 129.19, 127.89, 126.53, 121.49, 69.62, 69.48, 69.40, 54.17, 52.23, 51.14, 50.82, 37.13, 37.04, 36.79, 36.75, 36.72, 36.60, 32.86, 19.33, 19.01. 31P NMR (121 MHz, CDCl3) δ 18.57. HRMS calc. for C36H42N7O4PCl [M + H]+ 702.2724, found 702.2726. Synthesis of C16-conjugate of morpholinouridine [00627] Compound ELN0334-232) was synthesized according to Scheme 6.
Figure imgf000176_0002
Scheme 6
Figure imgf000177_0001
[00628] 2,2,2-trifluoroethyl 1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-2,4-dioxo- 1,2,3,4-tetrahydropyrimidine-5-carboxylate ELN0334-200: To a clear solution of 5-iodanyl-1-[6- (oxidanylmethyl)-4-[tri(phenyl)methyl]morpholin-2-yl]pyrimidine-2,4-dione (3 g, 5.04 mmol) in anhydrous ACN (25 mL) were added Et3N (2.55 g, 25.19 mmol, 3.51 mL) , CF3CH2OH (5.04 g, 50.38 mmol, 3.62 mL) and benzonitrile;bis(chloranyl)palladium-108 (96.63 mg, 251.92 μmol) . The reaction mixture was heated at 60°C in oil bath for 15 hr in CO atmosphere. The reaction mixture was filtered through celite pad and concentrated. The residue was re-dissolved in EtOAc, washed with water and brine. The organic layer was dried over Na2SO4, filtered and concentrated to get the crude product which was purified by silica gel column chromatography eluting with 30- 80 % EtOAc-hexane to afford ELN0334-200 (2.34 g, 3.93 mmol, 78 % yield) as white foam.1H NMR (600 MHz, CD3CN) δ 9.40 (s, 2H), 8.23 (s, 1H), 7.50 (s, 6H), 7.32 (t, J = 7.6 Hz, 6H), 7.22 (d, J = 7.5 Hz, 3H), 6.11 – 6.06 (m, 2H), 4.31 – 4.25 (m, 1H), 4.13 – 4.02 (m, 1H), 3.49 (q, J = 5.9 Hz, 3H), 3.41 (d, J = 11.5 Hz, 2H), 3.12 (dd, J = 12.0, 2.8 Hz, 2H), 2.94 (t, J = 6.0 Hz, 1H), 2.23 (s, 1H), 1.46 (t, J = 11.2 Hz, 1H), 1.21 (t, J = 7.1 Hz, 1H).13C NMR (151 MHz, CD3CN) δ 171.70, 161.91, 159.46, 149.88, 149.07, 130.24, 128.76, 127.50, 127.17, 125.34, 123.51, 121.67, 118.32, 104.11, 82.26, 78.53, 77.71, 63.39, 61.25, 61.02, 60.97, 60.78, 60.54, 53.64, 50.08, 21.16, 14.51, 1.73, 1.60, 1.46, 1.32, 1.18, 1.05, 0.91.19F NMR (565 MHz, CD3CN) δ -74.21.
Figure imgf000177_0002
[00629] N-hexadecyl-1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-2,4-dioxo- 1,2,3,4-tetrahydropyrimidine-5-carboxamide ELN0334-232: To a clear solution of 2,2,2- tris(fluoranyl)ethyl-2,4-bis(oxidanylidene)-1-[6-(oxidanylmethyl)-4- [tri(phenyl)methyl]morpholin-2-yl]pyrimidine-5-carboxylate (1.17 g, 1.97 mmol) in anhydrous ACN (15 mL) was added hexadecane-1-amine (1.42 g, 5.90 mmol) and the reaction mixture was allowed to stir at 50 ºC in oil bath for 12 hr. Then reaction mixture was concentrated to get the crude product which was purified by silica gel column chromatography eluting with 30-60 % EtOAc-hexane to afford ELN0334-232 (1.06 g, 1.44 mmol, 73 %) as white foam.1H NMR (600 MHz, CDCl3) δ 8.66 – 8.54 (m, 1H), 8.44 – 8.34 (m, 1H), 7.45 (s, 6H), 7.33 – 7.23 (m, 6H), 7.18 (d, J = 7.4 Hz, 3H), 6.16 – 6.09 (m, 1H), 4.31 – 4.24 (m, 1H), 3.59 (t, J = 5.7 Hz, 2H), 3.41 (dd, J = 11.0, 2.4 Hz, 1H), 3.38 – 3.26 (m, 2H), 3.07 (d, J = 11.7 Hz, 1H), 1.53 (dt, J = 7.5, 3.6 Hz, 3H), 1.47 – 1.41 (m, 1H), 1.24 (s, 26H), 0.90 – 0.86 (m, 3H).13C NMR (151 MHz, CDCl3) δ 162.69, 162.56, 161.62, 161.56, 149.07, 148.98, 145.99, 129.13, 127.99, 126.67, 106.15, 63.46, 63.43, 52.26, 48.63, 39.52, 39.50, 31.95, 29.73, 29.68, 29.66, 29.58, 29.46, 29.39, 29.36, 29.34, 27.05, 27.04, 22.72, 14.16. Synthesis of C22-conjugate of morpholinouridine [00630] Compound ELN0334-233 was synthesized according to Scheme 7.
Figure imgf000178_0001
[00631] N-docosyl-1-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-2,4-dioxo-1,2,3,4- tetrahydropyrimidine-5-carboxamide ELN0334-233:To a clear solution of 2,2,2- tris(fluoranyl)ethyl 2,4-bis(oxidanylidene)-1-[6-(oxidanylmethyl)-4- [tri(phenyl)methyl]morpholin-2-yl]pyrimidine-5-carboxylate (1.13 g, 1.90 mmol) in anhydrous ACN (15 mL) was added docosan-1-amine (1.87 g, 5.71 mmol) and the reaction mixture was allowed to stir at 55 ºC in oil bath for 12 hr. Then reaction mixture was concentrated to get the crude product which was purified by silica gel column chromatography eluting with 30-60 % EtOAc-hexane to afford ELN0334-233 (1.11 g, 1.35 mmol, 71 %) as white foam.1H NMR (600 MHz, CDCl3) δ 8.65 – 8.53 (m, 1H), 8.44 – 8.37 (m, 1H), 7.45 (s, 6H), 7.29 (t, J = 7.6 Hz, 6H), 7.19 (t, J = 7.5 Hz, 3H), 6.17 – 6.12 (m, 1H), 4.28 (ddd, J = 8.7, 4.6, 2.3 Hz, 1H), 3.60 (t, J = 4.6 Hz, 2H), 3.41 (dt, J = 11.3, 2.4 Hz, 1H), 3.33 (dt, J = 13.6, 7.3 Hz, 2H), 3.08 (dt, J = 11.9, 2.4 Hz, 1H), 1.53 (tt, J = 12.0, 5.7 Hz, 3H), 1.45 (td, J = 10.1, 2.5 Hz, 1H), 1.25 (d, J = 10.7 Hz, 39H), 0.88 (t, J = 7.0 Hz, 3H).13C NMR (151 MHz, CDCl3) δ 162.61, 161.65, 149.04, 146.09, 129.23, 128.11, 126.80, 106.31, 81.64, 78.28, 77.37, 77.16, 76.95, 76.91, 63.60, 63.57, 60.55, 53.56, 52.36, 48.74, 39.61, 32.06, 29.84, 29.80, 29.77, 29.69, 29.56, 29.50, 29.46, 27.15, 22.83, 14.26. Synthesis of C16-conjugate of piperidinouridine [00632] Compound ELN0334-235 was synthesized according to Scheme 8.
Figure imgf000179_0001
[00633] 1-((3R,5S)-5-(hydroxymethyl)-1-tritylpiperidin-3-yl)-5-iodopyrimidine-2,4(1H,3H)- dione ELN0334-199: To a clear solution of (1R)-1-[(1S,3R,5R)-5-(hydroxymethyl)-1- (triphenylmethyl)-3-piperidyl]pyrimidine-2,4-dione (1 g, 2.14 mmol) in DCM (15 mL) and MeOH (15 mL) was added K2CO3 (443.39 mg, 3.21 mmol) . Then the reaction mixture was cooled to 0 °C and ICl (1.04 g, 6.42 mmol) was added dropwise. The reaction mixture was allowed to stir at same temperature for 15 minutes. TLC showed formation of two products. Solvent was removed to get the crude products which were purified by silica gel column chromatography eluting with 0- 5 % MeOH-DCM to afford ELN0334-199 (721 mg, 1.21 mmol, 57 % yield) and (1R,5S,6R)-1- [(1S,3R,5S)-5-(hydroxymethyl)-1-(triphenylmethyl)-3-piperidyl]-5-iodo-6-methoxy- hexahydropyrimidine-2,4-dione (307 mg, 490.81 μmol, 23 % yield) as white foam. The second product was dissolved in 10 % Et3N-DCM and stirred at rt for 30 hr to convert to the 1st product (total yield is 80 %).1H NMR (600 MHz, CDCl3) δ 7.47 (s, 6H), 7.34 – 7.20 (m, 6H), 7.15 (s, 3H), 5.05 (tt, J = 11.4, 3.9 Hz, 1H), 3.48 (qd, J = 10.9, 5.9 Hz, 2H), 3.39 (d, J = 10.3 Hz, 1H), 3.32 – 3.27 (m, 1H), 2.35 (dq, J = 11.5, 5.9 Hz, 1H), 2.14 – 2.05 (m, 1H), 1.37 (t, J = 10.7 Hz, 1H), 1.20 (tt, J = 12.1, 6.9 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 160.15, 150.75, 145.37, 129.13, 127.86, 126.39, 77.40, 77.37, 77.16, 76.95, 68.19, 65.21, 53.56, 53.37, 53.21, 51.04, 38.84, 32.72.
Figure imgf000180_0001
[00634] 2,2,2-trifluoroethyl 1-((3R,5S)-5-(hydroxymethyl)-1-tritylpiperidin-3-yl)-2,4-dioxo- 1,2,3,4-tetrahydropyrimidine-5-carboxylate ELN0334-234: To a clear solution of (1R)-1- [(3S,5S)-5-(hydroxymethyl)-1-trityl-3-piperidyl]-5-iodo-pyrimidine-2,4-dione (782 mg, 1.32 mmol) in anhydrous ACN (9 mL) were added 2,2,2-trifluoroethanol (1.32 g, 13.18 mmol, 947.69 μL) , Et3N (666.70 mg, 6.59 mmol, 918.31 μL) and benzonitrile;bis(chloranyl)palladium-108 (25.27 mg, 65.89 μmol) . The reaction mixture was heated at 60 °C in oil bath for 15 hr in CO atmosphere. The reaction mixture was filtered through celite pad and concentrated. The residue was re-dissolved in EtOAc, washed with water and brine. The organic layer was dried over Na2SO4, filtered and concentrated to get the crude product which was purified by silica gel column chromatography eluting with 30-80 % EtOAc-hexane to afford ELN0334-234 (532 mg, 896.24 μmol, 68 % yield) as white foam.1H NMR (600 MHz, CDCl3) δ 7.47 (s, 6H), 7.34 – 7.20 (m, 6H), 7.15 (s, 3H), 5.07 (tt, J = 11.4, 3.9 Hz, 1H), 3.49 (qd, J = 10.9, 5.9 Hz, 2H), 3.41 (d, J = 10.3 Hz, 1H), 3.33 – 3.28 (m, 1H), 2.35 (dq, J = 11.5, 5.9 Hz, 1H), 2.14 – 2.05 (m, 1H), 1.37 (t, J = 10.7 Hz, 1H), 1.20 (tt, J = 12.1, 6.9 Hz, 2H).13C NMR (151 MHz, CDCl3) δ 160.15, 150.75, 145.37, 129.13, 127.86, 126.39, 77.40, 77.37, 77.16, 76.95, 68.19, 65.21,61.25, 61.02, 60.97, 60.78, 60.5453.56, 53.37, 53.21, 51.04, 38.84, 32.72.19F NMR (565 MHz, CD3CN) δ -73.81.
Figure imgf000181_0001
[00635] N-hexadecyl-1-((3R,5S)-5-(hydroxymethyl)-1-tritylpiperidin-3-yl)-2,4-dioxo- 1,2,3,4-tetrahydropyrimidine-5-carboxamide ELN0334-235: To a clear solution of 2,2,2- trifluoroethyl (1R)-1-[(3S,5S)-5-(hydroxymethyl)-1-trityl-3-piperidyl]-2,4-dioxo-pyrimidine-5- carboxylate (200 mg, 336.93 μmol) in anhydrous ACN (5 mL) was added hexadecan-1-amine (244.07 mg, 1.01 mmol) and the reaction mixture was allowed to stir at 55 °C in oil bath for 12 hr after which the solvent was removed to dryness and the solid residue was purified in silica gel colum chromatography eluting with 30-60 % EtOAc-hexane to afford ELN0334-235 (176 mg, 239.45 μmol, 71 % yield) as white foam.1H NMR (600 MHz, CDCl3) δ 8.64 – 8.52 (m, 1H), 8.43 – 8.35 (m, 1H), 7.42 (s, 6H), 7.31 – 7.23 (m, 6H), 7.17 (d, J = 7.4 Hz, 3H), 5.05 (tt, J = 11.4, 3.9 Hz, 1H), 3.48 (qd, J = 10.9, 5.9 Hz, 2H), 3.39 (d, J = 10.3 Hz, 1H), 3.32 – 3.27 (m, 1H), 2.35 (dq, J = 11.5, 5.9 Hz, 1H), 2.14 – 2.05 (m, 1H), 1.37 (t, J = 10.7 Hz, 1H), 1.20 (tt, J = 12.1, 6.9 Hz, 2H). 1.44 – 1.41 (m, 1H), 1.23 (s, 26H), 0.90 – 0.84 (m, 3H).13C NMR (151 MHz, CDCl3) δ 161.67, 161.53, 160.60, 160.53, 149.05, 148.96, 145.93, 129.13, 127.99, 126.66, 105.15, 62.86, 62.43, 52.22, 47.63, 38.72, 39.50, 31.95, 29.73, 29.68, 29.66, 29.58, 29.46, 29.39, 29.36, 29.34, 27.05, 27.04, 22.72, 14.16. Synthesis of C22-conjugate of piperidinouridine [00636] Compound ELN0334-236 was synthesized according to Scheme 9.
Figure imgf000182_0001
[00637] N-docosyl-1-((3R,5S)-5-(hydroxymethyl)-1-tritylpiperidin-3-yl)-2,4-dioxo-1,2,3,4- tetrahydropyrimidine-5-carboxamide ELN0334-236: To a clear solution of 2,2,2-trifluoroethyl (1R)-1-[(3S,5S)-5-(hydroxymethyl)-1-trityl-3-piperidyl]-2,4-dioxo-pyrimidine-5-carboxylate (243 mg, 409.37 μmol) in anhydrous ACN (5 mL) was added docosan-1-amine (399.89 mg, 1.23 mmol) and the reaction mixture was allowed to stir at 55 °C in oil bath for 16 hr after which the reaction mixture was concentrated to dryness. The solid residue was purified in silica gel column chromatography eluting with 20-45 % EtOAc-hexane to afford ELN0334-236 (225 mg, 274.67 μmol, 67 % yield) as white foam.1H NMR (600 MHz, CDCl3) δ 8.63 – 8.50 (m, 1H), 8.41 – 8.32 (m, 1H), 7.41 (s, 6H), 7.19 (t, J = 7.6 Hz, 6H), 7.09 (t, J = 7.5 Hz, 3H), 5.10 (m, 1H), 3.48 (qd, J = 10.9, 5.9 Hz, 2H), 3.40 (t, J = 4.6 Hz, 2H), 3.39 (dt, J = 11.3, 2.4 Hz, 1H), 2.39 (dt, J = 13.6, 7.3 Hz, 2H), 2.38 (dt, J = 11.9, 2.4 Hz, 1H), 1.51 (tt, J = 12.0, 5.7 Hz, 3H), 1.43 (td, J = 10.1, 2.5 Hz, 1H), 1.24 (d, J = 10.7 Hz, 39H), 0.87 (t, J = 7.0 Hz, 3H).13C NMR (151 MHz, CDCl3) δ 161.56, 161.42, 160.56, δ 160.51, 149.02, 146.07, 129.20, 128.11, 126.80, 105.31, 81.60, 78.28, 77.37, 77.16, 76.95, 76.91, 62.89, 61.53, 60.55, 53.56, 52.36, 48.74, 39.61, 32.06, 29.84, 29.80, 29.77, 29.69, 29.56, 29.50, 29.46, 27.15, 22.83, 14.26. Synthesis of 2-F-6-aminopurine PMO phosphoramidite [00638] Compound 103 is synthesized according to Scheme 10.
Figure imgf000183_0001
Figure imgf000183_0004
102
2-cyanoethyl N,N,N',N'- tetraisopropylphosphorodiamidite X - O, CH2, NR, S where R is 5-(ethylthio)-1 H-tetrazole protecting group or alkyl or ligand
DCM, rt, 0.5 h
Figure imgf000183_0002
103
Scheme 10: 2-F-6-aminopurine PMO phosphoramidite synthesis as precursor of diaminopurine (DAP) and C2-conjugation
Synthesis of BisPOM-Morpholino-VP phosphoramidite
[00639] Compound ELN0132-972 was synthesized according to Scheme 11.
Figure imgf000183_0003
Figure imgf000183_0005
ELN0132-186
ELN0132-605 ELN0132-606
Figure imgf000183_0006
ELN0132-972
Scheme 11 Synthesis of Morpholino VP phosphoramidite [00640] Compound ELN0132-972 was synthesized as follows:
Figure imgf000184_0001
[00641] (2S,6R)-6-(2,4-dioxopyrimidin-1-yl)-4-trityl-morpholine-2-carbaldehyde ELN0132- 673: To a clear solution of 1-[(2R,6S)-6-(hydroxymethyl)-4-trityl-morpholin-2-yl]pyrimidine-2,4- dione (0.7 g, 1.49 mmol) in DCM (20 mL) was added Dess-Martin Periodinane (790.41 mg, 1.86 mmol) at O °C. The resulting mixture was stirred for 4 hr at 22 °C and then cooled again to 0 °C. The reaction mixture was diluted with NaHCO3 solution (25 mL). The organic layer was separated and washed with 5% sodium thiosulfate solution (25 mL). The resulting organic layer was separated, dried over anhydrous Na2SO4, filtered and filtrate was evaporated to dryness to afford white solid (0.67 g, 96% yield) which was used for next sept without further purification.1H NMR (600 MHz, CDCl3) δ 9.54 (s, 1H), 9.16 (s, 1H), 7.46 (s, 12H), 7.34 – 7.15 (m, 16H), 6.25 (dd, J = 9.7, 2.3 Hz, 1H), 5.70 – 5.63 (m, 1H), 4.61 (dd, J = 11.0, 2.8 Hz, 1H), 3.64 – 3.29 (m, 3H), 1.55 – 1.38 (m, 2H) ppm.
Figure imgf000184_0002
trityl-morpholin-2-yl]vinyl]phosphoryl]oxymethyl 2,2-dimethylpropanoate ELN0132-966: Sodium hydride (462.02 mg, 11.55 mmol, 60% purity) was suspended in anhydrous THF (30 mL) and cooled to -78°C. VP reagent [bis(2,2-dimethylpropanoyloxymethoxy)phosphorylmethyl- (2,2-dimethylpropanoyloxymethoxy) phosphoryl]oxymethyl-2,2-dimethylpropanoate (6.09 g, 9.63 mmol) was added to this suspension and the resulting mixture was stirred for 20 min at the same temperature. The crude aldehyde obtained from previous reaction (ELN0132-673) was dissolved in anhydrous THF (30 mL) and transferred dropwise to the previous flask and the resulting mixture was stirred for 20 min at -78°C. The ice bath was removed, and the reaction mixture was stirred for 2 hr at rt. The crude reaction mixture was quenched with saturated ammonium chloride solution, and the product was extracted into ethyl acetate (50 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated to dryness under reduced pressure. The crude residue thus obtained was purified by flash column chromatography (gradient: 10-60% EtOAc in hexanes) to afford ELN0132-966 (1.78 g, 2.30 mmol, 60% yield) as mixed with the VP reagent.1H NMR (600 MHz, DMSO) δ 11.34 (s, 1H), 7.60 – 7.28 (m, 14H), 7.20 (s, 3H), 6.71 – 6.51 (m, 1H), 6.10 (dd, J = 9.6, 2.4 Hz, 1H), 6.06 – 5.96 (m, 1H), 5.66 – 5.47 (m, 9H), 4.97 (d, J = 10.2 Hz, 1H), 3.23 – 3.12 (m, 2H), 3.11 – 3.05 (m, 1H), 1.17 (s, 20H), 1.06 (d, J = 2.1 Hz, 18H) ppm. 13C NMR (151 MHz, DMSO) δ 176.08, 162.92, 149.84, 148.47, 148.43, 140.36, 128.88, 127.90, 126.40, 117.71, 116.47, 101.80, 81.78, 81.76, 81.74, 81.51, 81.47, 81.43, 79.78, 76.32, 74.70, 74.56, 59.77, 51.20, 50.82, 38.20, 38.13, 38.12, 26.45, 26.40 ppm.31P NMR (243 MHz, DMSO) δ 18.85, 17.99 ppm.
Figure imgf000185_0001
[00643] [[(E)-2-[(2R,6R)-6-[2,4-bis(oxidanylidene)pyrimidin-1-yl]morpholin-2-yl]vinyl]- [2,2-di(methyl)propanoyloxymethoxy]phosphoryl]oxymethyl 2,2-di(methyl)propanoate ELN0132-971: To a clear solution of ELN0132-966 (1.0 g, 1.29 mmol) in trifluoro ethanol (5 mL) was added formic acid (5 mL) in single portion at 0°C and then ice bath was removed. Stirring continued for 0.5 hr and TLC was checked which indicated consumption of the starting material. All the volatile matters were removed under high vacuum pump and the residue thus obtained was purified by flash column chromatography (gradient: 0-10% MeOH in DCM) to afford ELN0132- 971 (0.51 g, 74% yield) as hygroscopic foam.1H NMR (600 MHz, DMSO) δ 11.40 (s, 1H), 7.74 (d, J = 8.1 Hz, 1H), 6.81 – 6.58 (m, 1H), 6.05 – 5.95 (m, 1H), 5.66 – 5.55 (m, 6H), 4.41 – 4.30 (m, 1H), 2.96 (dd, J = 12.7, 2.6 Hz, 1H), 2.86 (dd, J = 12.6, 2.7 Hz, 1H), 2.66 (dd, J = 12.6, 10.2 Hz, 1H), 2.28 (dd, J = 12.9, 10.5 Hz, 1H), 1.14 (d, J = 14.6 Hz, 18H) ppm.13C NMR (151 MHz, DMSO) δ 176.03, 176.01, 162.95, 150.11, 149.24, 149.20, 140.93, 116.69, 115.45, 101.83, 81.53, 81.49, 81.46, 79.66, 77.10, 76.96, 54.94, 48.06, 48.05, 47.42, 38.20, 38.17, 26.48, 26.43 ppm. 31P NMR (243 MHz, DMSO) δ 18.27 ppm.
Figure imgf000186_0001
[00644] Minor isomer isolated at this stage (Z-VP) Data: 1H NMR (600 MHz, DMSO) δ 11.54 – 11.40 (m, 1H), 8.12 (d, J = 11.0 Hz, 1H), 7.81 (dd, J = 8.1, 6.9 Hz, 1H), 6.82 – 6.60 (m, 1H), 6.18 – 6.07 (m, 1H), 5.78 – 5.66 (m, 2H), 5.65 – 5.55 (m, 5H), 4.67 – 4.41 (m, 1H), 4.38 – 4.19 (m, 1H), 3.96 – 3.80 (m, 1H), 3.37 (dd, J = 13.1, 10.5 Hz, 1H), 3.00 – 2.88 (m, 1H), 2.57 – 2.51 (m, 1H), 1.14 (d, J = 1.8 Hz, 9H), 1.12 (d, J = 0.8 Hz, 9H) ppm.13C NMR (151 MHz, DMSO) δ 176.04, 176.02, 162.92, 162.87, 161.43, 161.40, 149.96, 147.63, 147.58, 147.34, 147.30, 140.68, 140.61, 118.52, 118.43, 117.28, 117.19, 102.29, 102.27, 81.58, 81.55, 81.51, 78.71, 77.89, 75.52, 75.38, 74.54, 74.40, 47.05, 46.37, 41.69, 41.27, 38.21, 38.18, 26.48, 26.44 ppm. 31P NMR (243 MHz, DMSO) δ 17.36.
Figure imgf000186_0002
[2,4-bis(oxi danylidene)pyrimidin-1-yl]morpholin-2-yl]vinyl]-[2,2- di(methyl)propanoyloxymethoxy]phos phoryl]oxymethyl-2,2-di(methyl)propanoate ELN0132- 972: Compound ELN0132-971 (1.0 g, 1.88 mmol) was dissolved in dry DCM (15 mL). To the clear solution was added 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (311.91 mg, 1.03 mmol, 328.67 μL) and 5-(ethylthio)-1H-tetrazole (61.23 mg, 470.37 μmol) in single portions and stirred at for 0.5 hr. All the volatile matters were removed under high vacuum pump and crude residue was purified by flash chromatography (70-90% EtOAc in hexane) to afford ELN0132-972 (0.58 g, 42% yield) as white hygroscopic foam.1H NMR (600 MHz, CD3CN) δ 8.98 (s, 1H), 7.59 (dd, J = 8.1, 7.1 Hz, 1H), 6.81 – 6.64 (m, 1H), 6.13 – 6.02 (m, 1H), 5.69 – 5.56 (m, 6H), 4.46 – 4.34 (m, 1H), 3.83 (qdd, J = 6.1, 3.9, 0.9 Hz, 2H), 3.58 – 3.43 (m, 2H), 3.35 – 3.25 (m, 0H), 2.68 (td, J = 6.0, 2.9 Hz, 2H), 2.57 – 2.48 (m, 1H), 2.46 – 2.32 (m, 1H), 1.19 – 1.16 (m, 24H), 1.13 (d, J = 6.8 Hz, 6H) ppm.13C NMR (151 MHz, CD3CN) δ 177.54, 163.69, 163.67, 150.89, 150.86, 149.45, 149.41, 141.46, 141.37, 119.67, 119.55, 117.51, 117.47, 102.70, 102.65, 82.68, 82.65, 82.61, 81.25, 81.21, 81.09, 81.01, 77.82, 77.78, 77.75, 77.68, 77.64, 77.60, 77.54, 61.12, 61.07, 60.96, 60.91, 49.22, 49.07, 49.04, 48.87, 47.82, 47.78, 47.71, 44.70, 44.62, 44.58, 44.50, 39.39,
39.36, 27.11, 27.06, 24.81, 24.76, 24.74, 24.72, 24.68, 23.16, 23.15, 23.07, 21.96, 21.14, 20.95, 20.92, 20.90, 20.87 ppm. 31P NMR (243 MHz, CD3CN) 5 127.12, 125.09, 17.73 ppm.
Table 3A: Exemplary siRNAs sequences targeting mouse-TTR sequence for in vitro / in vivo evaluation with car-Morpholino-U, C, A and G (Piperidine Nucleosides) (PS sequences)
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Table 3B: Exemplary siRNAs sequences targeting mouse-TTR sequence for in vitro / in vivo evaluation with car-Morpholino-U, C, A and G (Piperidine Nucleosides) (PO sequences)
Figure imgf000189_0002
Figure imgf000190_0001
Figure imgf000191_0001
Table 4A: Exemplary siRNA sequences targeting mouse-TTR sequence for in vitro / in vivo evaluation with Morpholino-U, C, A and G (Morpholino-PS sequences)
Figure imgf000191_0002
Figure imgf000192_0001
Figure imgf000193_0001
Table 4B: Exemplary siRNA sequences targeting mouse-TTR sequence for in vitro / in vivo evaluation with Morpholino-U, C, A and G (Morpholino-PO sequences)
Figure imgf000193_0002
Figure imgf000194_0001
Figure imgf000195_0001
[00646] The analogous thiomorphino or piperazinyl duplex sequence to any one of Sequences 1 - 95 shown in Tables 4A and 4B are provided by making the following substitutions (Table 4C) and each are expressly contemplated by this disclosure:
Table 4C:
Figure imgf000195_0002
Figure imgf000196_0002
[00647] Exemplary control/reference sequences with GNA and TNA at Position 7 for off-target mitigation evaluation are shown in FIG. 13.
Morpholino .phosphor amidites:
[00648] Chimeric oligonucleotides with the morpholino phosphoramidate were reported by Jiang et. al.1 Later RNA interference in mammalian cells by siRNAs modified with morpholino nucleoside analogues were demonstrated by the same group2. Later the phosphorothioate linked backbone were introduced from Marvin Caruthers’ laboratory and described as Thiophosphoramidate Morpholino Oligonucleotides (TMOs)3. Recently, Caruthers and Veedu published the thiomorpholinos oligonucleotides and described as a robust class of next generation platforms for alternate mRNA splicing.4 Veedu published (MNA)-uridine phosphoramidite as a mixmer antisense oligonucleotide.5 Exemplary morpholino amidites shown here (I-IV and Y271- Y274, FIG. 14) were synthesized following the literature procedure. The CPG L437 was synsthesized according to Scheme 5.
Figure imgf000196_0001
Scheme 5: Synthesis of CPG L437
[00649] l-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy- morpholin-2-yl]pyrimidine-2, 4-dione Compound 6: Commercially available compound 5 (4.0 g, 7.32 mmol)was dissolved in methanol (40 mL) at 22 °C and to this reaction mixture was added sodium (meta)periodate (2.35 g, 10.98 mmol) and water (5 mL) . Reaction miture was stirred for 2 hr at the same temperature. The solvent was evaporated under vacuum, and the residue was taken in water (20 mL) and extracted with EtOAc (3 x 25 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum to yield a white foam. [00650] To an ice cooled solution of hydroxylamine hydrochloride (2.54 g, 36.59 mmol) in MeOH/H2O (1:9, v/v, 36 mL), sodium acetate (3.00 g, 36.59 mmol) was slowly added. After 5 min, a solution of dialdehyde prepared in previous step in MeOH (30 mL) was slowly added, and the reaction mixture was stirred for 16 hr at 22 °C. The solvent was evaporated under vacuum, and water was added to the resulting residue (100 mL) and extracted with EtOAc (3 x 25 mL). The combined organic phase was dried over Na2SO4 and evaporated under vacuum. The crude residue was purified by flash column chromatography (gradient: 0–10% MeOH in CH2Cl2) to yield the dioxime (mixture of regio and stereo isomers). [00651] To an ice cooled solution of dioxime in anhydrous MeOH (20 mL), borane pyridine complex 8M (1.06 g, 76.84 mmol, 1.16 mL) was added dropwise. After 5 min of stirring at 10 °C, hydrogen chloride solution 4.0M in dioxane (453.61 mg, 12.44 mmol, 567.01 μL) was added slowly to adjust pH = 4.5–5. Then, the reaction mixture was stirred at 22 °C for 2 hr. The reaction was quenched with a solution of 10% aqueous NaOH saturated with NaCl, and the reaction mixture pH was adjusted to 8. Then, the product was extracted with EtOAc (100 mL). The organic phase was dried over Na2SO4, evaporated under vacuum, and purified by flash column chromatography (gradient: 0-5% MeOH in DCM) to afford (1.1 g, 28% yield) as white foam. 1H NMR (600 MHz, DMSO) δ 11.36 (d, J = 2.2 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.40 – 7.35 (m, 2H), 7.32 (dd, J = 8.5, 7.0 Hz, 2H), 7.24 (dt, J = 8.6, 3.1 Hz, 5H), 6.93 – 6.86 (m, 4H), 5.74 (d, J = 3.4 Hz, 1H), 5.50 (d, J = 4.8 Hz, 1H), 5.31 (dd, J = 8.1, 2.2 Hz, 1H), 5.15 (d, J = 5.6 Hz, 1H), 4.08 (h, J = 5.2 Hz, 2H), 3.95 (td, J = 4.7, 2.7 Hz, 1H), 3.74 (s, 6H), 3.28 – 3.19 (m, 2H) ppm. 13C NMR (151 MHz, DMSO) δ 163.0, 158.1, 158.1, 150.5, 144.7, 140.6, 135.4, 135.2, 129.8, 127.9, 127.7, 126.8, 113.3, 113.3, 101.5, 88.9, 85.8, 82.4, 73.4, 69.6, 63.0, 55.1 ppm. [00652] 4-[(2S,6R)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-(2,4- dioxopyrimidin-1-yl)morpholin-4-yl]oxy-4-oxo-butanoic acid Compound 75: To a clear solution of 1-[(2R,6S)-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-4-hydroxy-morpholin-2- yl]pyrimidine-2,4-dione (0.19 g, 348.25 μmol) in DCM (10 mL) was added DMAP (127.64 mg, 1.04 mmol) and succinic anhydride (69.70 mg, 696.50 μmol) in single portion. Reaction mixture was stirred at 22 °C for 2 hr. To this reaction mixture was added DCM (10 mL) and washed with 10% NH4Cl solution (2 x 10 mL). Organic layer was separated, dried over anhydrous Na2SO4, filtered and the filtrate was evaporated to dryness. Crude compound was purified by flash column chromatography (gradient: 5-10% MeOH in DCM) to afford 7 (0.16 g, 247.81 μmol, 71% yield) as white foam.1H NMR (600 MHz, DMSO) δ 12.25 (s, 1H), 11.46 (d, J = 2.2 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.31 (t, J = 7.8 Hz, 2H), 7.27 – 7.19 (m, 5H), 6.92 – 6.82 (m, 4H), 5.70 (d, J = 8.1 Hz, 1H), 4.15 – 3.97 (m, 1H), 3.73 (d, J = 0.9 Hz, 6H), 3.33 (s, 2H), 3.17 (d, J = 5.0 Hz, 1H), 3.12 (s, 1H), 3.04 (s, 1H), 2.90 (s, 1H), 2.55 – 2.51 (m, 3H), 2.49 – 2.45 (m, 1H) ppm.13C NMR (151 MHz, DMSO) δ 173.4, 173.3, 173.1, 172.6, 170.3, 162.9, 158.1, 149.9, 144.7, 140.7, 135.3, 129.7, 127.9, 127.7, 127.4, 126.7, 113.2, 112.8, 102.2, 85.6, 55.0, 51.4, 48.6, 28.8, 28.6, 28.6, 28.5, 27.4 ppm. [00653] Preparation of CPG L437: Added 7 (0.15 g, 232.32 μmol) and diisopropylethylamine (120.10 mg, 929.29 μmol, 161.86 μL) into rb flask. Then added dry ACN (20 mL). Stirred well to dissolve and then HBTU (88.11 mg, 232.32 μmol) to preactivate acid. Let stirr for ~5 min, then added CPG. Capped and put on mechanical shaker overnight. Next day filtered CPG, washed with ACN, then MeOH, then ACN, then diethyl ether. Dried for ~ 5 minutes, then transferred back to rb flask for capping. Added 30% acetic anhydride in pyridine (50ml total) and 1% TEA. Capped and put back on mechanical shaker for 3 hours. After 3 hours took off and washed CPG as follows: 10% H2O/THF, then MeOH, then 10% H2O/THF, then MeOH, then ACN, the ether (~250 mL each solvent for washing). Transferred to rb flask and dried CPG in high vacuum overnight. [00654] Checking the Loading: Weighted out 56 mg and loaded into 250ml volumetric flask. Then added 0.1M toluene-p-sulfonic acid in ACN up to measure line. Sonicated and settled for 1 hour. Checked loading by spectrophotometer and Beers law. Measured solution into UV cuvette and measured UV absorbance at 411 nm. Check worksheet for raw data. Calculated loading using beers law = [250 (mL) x (absorbance A) x 35.5 (extinction coefficient of DMTr)] /weight of CPG (mg). Yield: 1.04 g, Loading: 92 µmol/g. [00655] Amidite Y274 and CPG L437 were incorporated into oligonucleotides shown in Table 5 for testing the nuclease resistance abilities of these modifications. Table 5: Exemplary oligonucleotides comprising monomers Y274 and L437.
Figure imgf000198_0001
References
1. Zhang, N.; Tan, C.; Cai, P.; Jiang, Y.; Zhang, P.; Zhao, Y., Synthesis and properties of morpholino chimeric oligonucleotides. Tetrahedron Lett. 2008, 49, 3570-3573.
2. Zhang, N.; Tan, C.; Cai, P.; Zhang, P.; Zhao, Y.; Jiang, Y., RNA interference in mammalian cells by siRNAs modified with morpholino nucleoside analogues. Bioorganic & Medicinal Chemistry 2009, 17, 2441-2446.
3. Langner, H. K.; Jastrzebska, K.; Caruthers, M. H., Synthesis and Characterization of Thiophosphorami date Morpholino Oligonucleotides and Chimeras. Journal of the American Chemical Society 2020, 142, 16240-16253.
4. Le, B. T.; Paul, S.; Jastrzebska, K.; Langer, H.; Caruthers, M. H.; Veedu, R. N., Thiomorpholino oligonucleotides as a robust class of next generation platforms for alternate mRNA splicing. Proceedings of the National Academy of Sciences 2022, 119, e2207956119.
5. Chen, S.; Le, B. T.; Rahimizadeh, K.; Shaikh, K.; Mohal, N.; Veedu, R. N., Synthesis of a Morpholino Nucleic Acid (MNA)-Uridine Phosphorami dite, and Exon Skipping Using MNA/2'-O-Methyl Mixmer Antisense Oligonucleotide. In Molecules2016; Vol. 21.
New sequences with morpholino nucleotides:
1) Homo oligomers a) Oligonucleotides (ONs) composed of only Morpholino phosphoramidites. b) Oligonucleotides (ONs) composed of only Piperidino phosphoramidites.. c) Oligonucleotides (ONs) composed of both Morpholino phosphoramidites and Piperidino phosphoramidites..
2) Chimeric oligomers a) ONs Composed of Morpholino or Piperidino amidites and all other modified nucleosides viz., MOE, NMA, 2’OMe, 2’F, b) Ligand conjugated nucleosides (modified sugar, base, backbone), C5-modified pyrimidines, C8-, N2- and N6-modified purines.
3) Multiple targeting ONs with two different flavors: a) Both sequences have morpholino or Piperidino building blocks (for Splice modulation) b) Chimeric dual targeting sequences i) First one is RNase H antisense strand, antisense strand of siRNA, miRNA mimic antisense strand, antogomirs with known 2’-/ other modifications or fully modified SSOs. ii) Second one for splice modulation containing morpholino or piperidino building blocks. c) Both aforementioned classes will have one or more conjugation site from 1.
Morpholino/Piperidino or 2. Modified nucleosides.
Example 2: In vivo evaluation of exemplary siRNAs [00656] Evaluation of silencing in mice. All studies are conducted using protocols consistent with local, state, and federal regulations, as applicable, and approved by the Institutional Animal Care and Use Committee at Alnylam Pharmaceuticals. Mice receive a single subscapular subcutaneous injection of 1 mg/kg siRNA (e.g., an siRNA from Table 4A or 4B), prepared in an injection volume of 10 pL/g body weight in PBS. At the indicated time pre- or post-dosing, animals are anesthetized with isofluorane and blood is obtained via retroorbital bleed. TTR protein is quantified by ELISA from serum isolated from whole blood. The ELISA is performed according to the manufacturer’s protocol (ALPCO, 41-PALMS-E01) after a 3025-fold dilution of the serum samples. Data are normalized to prebleed TTR levels for each individual. All samples are assayed in duplicate, and each data point is the average of all the mice within each cohort (n = 3). Data are analyzed using a two-way ANOVA with a Tukey post-hoc test for multiple comparison in GraphPad Prism.
[00657] Luciferase assay. COS-7 cells are cultured at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells are co-transfected in 96-well plates (15,000 cells/well) with 10 ng luciferase reporter plasmid and 0.64 pM to 50 nM siRNA in 5-fold dilutions using 2 pg ml Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. Cells are harvested 48 h after transfection for the dual luciferase assay (Promega) according to the manufacturer’s instructions. The on-target and off-target reporters are generated by Blue Heron Biotech by cloning into the psiCHECK2 vector between Xhol and Notl restriction sites in the 3'-UTR of Renilla luciferase. The on-target reporter plasmid contained a single site perfectly complementary to the guide strand in the 3'-UTR of Renilla luciferase (5'-AAAACAGTGTTCTTGCTCTATAA-3', SEQ ID NO: 234). The off-target reporter plasmid contained four tandem seed-complementary sites (5'-GCTCTATAA-3') separated by a 19-nucleotide spacer (5'-TAATATTACATAAATAAAA-3' SEQ ID NO: 236) in the 3'-UTR of Renilla luciferase.1'5 Both the on-target and off-target regions are flanked at the 5’ ends by 5'-ATAAACAAGGTTTGACATCAATCTAGCTATATCTTTAAGAATGATAAACT- 3' (SEQ ID NO: 237) and at the 3' ends by 5'- GACATTGGTGAGGAAAAATCCTTTGGCCGTTTCCAAGATCTGACAGTGCA-3' (SEQ ID NO: 238). Both plasmids co-expressed firefly luciferase as a transfection control. The luciferase measurements are conducted as reported previously6.
[00658] RNA-seq analysis. Primary rat hepatocytes (BioIVT) are seeded in 96-well collagen Lcoated plates (Gibco) at approximately 50,000 cells/well in 95 pL INVITROGRO CP Rodent Medium (BioIVT). Lipid/siRNA complex (0.25 pL RNAiMax (Thermo Fisher Scientific) and 1 pL siRNA in 3.75 pL Opti-MEM incubated for 15 min) is added to cells and incubated for 48 h at 37 °C. The final concentration of the siRNA is 50 nM. Experiments are performed in quadruplicate. The media is removed after 48 h, and RNA is extracted using the miRNeasy 96 kit (Qiagen). The cDNA libraries are prepared with the TruSeq Stranded Total RNA Library Prep Kit (Illumina) and sequenced on the HiSeq or NextSeq500 sequencers (Illumina), all according to manufacturer’s instructions. Raw RNA-seq reads are filtered with minimal mean quality scores of 25 and minimal remaining length of 36 using fastq-mcf. Filtered reads are aligned to the Rattus norvegicus genome (Rnor_6.0) using STAR with default parameters. Uniquely aligned reads are counted using featureCounts7. Differential gene expression analysis is performed using the R package DESeq28. [00659] Si-1 shown in Table 4A and/or the siRNAs shown in Table 6 are used as control/comparison.
Table 6: Contol/comparative duplexes for the in vivo study
Figure imgf000201_0001
REFERENCES
1. Bramsen, J. B.; Pakula, M. M.; Hansen, T. B.; Bus, C.; Langkjaer, N.; Odadzic, D.; Smicius, R.; Wengel, S. L.; Chattopadhyaya, J.; Engels, J. W.; Herdewijn, P.; Wengel, J.; Kjems, J., A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects. Nucleic Acids Res. 2010, 38 (17), 5761- 5773.
2. Vaish, N.; Chen, F.; Seth, S.; Fosnaugh, K.; Liu, Y.; Adami, R.; Brown, T.; Chen, Y.; Harvie, P.; Johns, R.; Severson, G.; Granger, B.; Charmley, P.; Houston, M.; Templin, M. V.; Polisky, B., Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs. Nucleic Acids Res. 2010, 39 (5), 1823-1832.
3. Ui-Tei, K.; Naito, Y.; Nishi, K.; Juni, A.; Saigo, K., Thermodynamic stability and Watson- Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA- based off-target effect. Nucleic Acids Res. 2008, 36 (22), 7100-7109.
4. Lee, H.-S.; Seok, H.; Lee, D. H.; Ham, J.; Lee, W.; Youm, E. M.; Yoo, J. S.; Lee, Y.-S.; Jang, E.-S.; Chi, S. W., Abasic pivot substitution harnesses target specificity of RNA interference. Nat. Commun. 2015, 6 (1), 10154.
5. Doench, J. G; Petersen, C. P.; Sharp, P. A., siRNAs can function as miRNAs. Genes Dev 2003, 77 (4), 438-442.
6. Akabane-Nakata, M.; Erande, N. D.; Kumar, P.; Degaonkar, R.; Gilbert, J. A.; Qin, J.; Mendez, M.; Woods, L. B.; Jiang, Y.; Janas, Maja M.; O’Flaherty, D. K.; Zlatev, I.; Schlegel, Mark K.; Matsuda, S.; Egli, M.; Manoharan, M., siRNAs containing 2'- fluorinated Northem-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs. Nucleic Acids Res. 2021, 49 (5), 2435-2449.
7. Liao, Y.; Smyth, G. K.; Shi, W., featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014, 30 (7), 923-930.
8. Love, M. I.; Huber, W.; Anders, S., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 75 (12), 550. Example 3: Synthesis of 5’-S-morpholino monomers
[00660] 5'-S-morpholino uridine monomer (S-106) was synthesized according to Scheme 12.
Figure imgf000202_0001
[00661] l-((2R,3R,4S,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,4- dihydroxytetrahydrothiophen-2-yl)pyrimidine-2,4(lH,3H)-dione (S-102): To a clear solution of (lS)-l-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]pyrimidine- 2,4-dione (1 g, 3.84 mmol) in anhydrous DMF (10 mL) were added imidazole (653.95 mg, 9.61 mmol) and TBDPSC1 (723.88 mg, 4.80 mmol) and the reaction mixture was allowed to stir at rt for 6 hr. Solvent was removed in reduced pressure and dried properly to get the solid residue which was triturated with 50 % EtOAc-Hexane 4 times to get (lS)-l-[(2R,3R,4S,5R)-5-[[l,l- dimethylethyl(diphenyl)silyl]oxymethyl]-3,4-dihydroxy-tetrahydrothiophen-2-yl]pyrimidine-2,4- dione (1.75 g, 3.51 mmol, 91.34% yield) as gummy solid which was used for the next step without any further purification. LCMS was used for characterization.
Figure imgf000203_0001
S-103 [00662] 1-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)thiomorpholin-2-yl)pyrimidine- 2,4(1H,3H)-dione (S-103): To a clear solution of (1S)-1-[(2R,3R,4S,5R)-5-[[1,1- dimethylethyl(dimethyl)silyl]oxymethyl]-3,4-dihydroxy-tetrahydrothiophen-2-yl]pyrimidine-2,4- dione (1 g, 2.67 mmol) in 80 % MeOH-DCM (50 mL) were added NaIO4 (714.23 mg, 3.34 mmol) and (NH4)2B4O7.4H2O (1.76 g, 6.68 mmol) and the reaction mixture was allowed to stir at rt for 1.5 hr. Solvent was filtered through celite pad and to the filtrate were added NaBH3CN (335.58 mg, 5.34 mmol) , MS(4 A°, 300 mg/mmol) and AcOH (320.68 mg, 5.34 mmol, 305.41 μL). The reaction mixture was allowed to stir for 2 hr. Solvent was filtered through celite pad and the filtrate was removed in reduced pressure to get the solid crude residue which was purified by silica gel column chromatography eluting with 0-10 % MeOH-DCM to afford (1S)-1-[(2R,6S)-6- [[1,1-dimethylethyl(dimethyl)silyl] oxymethyl]thiomorpholin-2-yl]pyrimidine-2,4-dione (129 mg, 360.80 μmol, 13.51% yield) as white solid.1H NMR (600 MHz, CDCl3) δ 7.80 (dd, J = 8.1, 1.0 Hz, 1H), 5.76 (d, J = 8.1 Hz, 1H), 5.65 (dd, J = 8.8, 3.5 Hz, 1H), 3.69 (dd, J = 10.3, 7.7 Hz, 1H), 3.61 (dd, J = 10.3, 5.6 Hz, 1H), 3.44 (dd, J = 12.7, 3.5 Hz, 1H), 3.33 (dd, J = 13.0, 2.9 Hz, 1H), 3.20 (tdd, J = 8.4, 5.6, 2.8 Hz, 1H), 3.02 (dd, J = 12.8, 8.7 Hz, 1H), 2.82 (dd, J = 13.0, 8.8 Hz, 1H), 0.88 (s, 9H), 0.05 (d, J = 1.6 Hz, 6H).13C NMR (151 MHz, CDCl3) δ 163.43, 150.61, 141.32, 103.00, 64.24, 52.55, 51.56, 48.51, 45.23, 25.95, 18.41, -5.25, -5.29.
Figure imgf000203_0002
[00663] 1-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylthiomorpholin-2- yl)pyrimidine-2,4(1H,3H)-dione (S-104): To a clear solution of (1S)-1-[(2R,6S)-6-[[1,1- dimethylethyl(dimethyl)silyl]oxymethyl]thiomorpholin-2-yl]pyrimidine-2,4-dione (97 mg, 271.30 μmol) in anhydrous DMF (6 mL) were added Et3N (82.36 mg, 813.89 μmol, 113.44 μL) and Ph3CCl (113.45 mg, 406.94 μmol) and the reaction mixture was allowed to stir at rt for 3 hr. The reaction mixture was diluted with EtOAc, washed with water 5 times to remove DMF followed by brine wash. The organic layer was dried over Na2SO4, filtered and concentrated in reduced pressure to get the crude residue which was purified by silica gel column chromatography eluting with 30-70 % EtOAC-Hexane to afford (1S)-1-[(2R,4S,6S)-6-[[1,1- dimethylethyl(dimethyl)silyl]oxymethyl]-4-(triphenylmethyl)thiomorpholin-2-yl]pyrimidine-2,4- dione (148 mg, 246.73 μmol, 90.94% yield) as white foam.1H NMR (600 MHz, CDCl3) δ 9.42 – 9.35 (m, 1H), 7.53 (s, 5H), 7.43 – 7.35 (m, 7H), 7.29 – 7.22 (m, 3H), 6.34 (dt, J = 10.5, 3.0 Hz, 1H), 5.72 (dd, J = 8.1, 2.0 Hz, 1H), 3.82 (dtt, J = 8.0, 5.8, 2.8 Hz, 1H), 3.74 (d, J = 12.2 Hz, 1H), 3.65 (dt, J = 10.4, 5.6 Hz, 2H), 3.61 – 3.54 (m, 1H), 1.74 (t, J = 11.1 Hz, 1H), 1.45 (t, J = 11.4 Hz, 1H), 0.88 (s, 9H), 0.11 – 0.04 (m, 6H).13C NMR (151 MHz, CDCl3) δ 162.84, 150.16, 140.77, 129.92, 129.12, 128.15, 128.05, 126.61, 102.93, 78.17, 77.37, 77.16, 76.95, 63.90, 56.39, 53.76, 53.42, 45.31, 25.81, -5.28, -5.42.
Figure imgf000204_0001
[00664] 1-((2R,6S)-6-(hydroxymethyl)-4-tritylthiomorpholin-2-yl)pyrimidine-2,4(1H,3H)- dione (S-105): To a clear solution of (1S)-1-[(2R,4S,6S)-6-[[1,1- dimethylethyl(dimethyl)silyl]oxymethyl]-4-(triphenylmethyl)thiomorpholin-2-yl]pyrimidine-2,4- dione (121 mg, 201.71 μmol) in anhydrous THF (5 mL) was added 1 M TBAF (in THF) (0.3 mL, 301.50 μmol) and the reaction mixture was allowed to stir at rt for 2 hr. Solvent was removed in reduced pressure to get the crude product which was purified by silica gel column chromatography eluting with 0-7 % MeOH-DCM to afford (1S)-1-[(2R,4S,6S)-6-(hydroxymethyl)-4- (triphenylmethyl)thiomorpholin-2-yl]pyrimidine-2,4-dione (91 mg, 187.40 μmol, 92.90% yield) as white foam. Due to solubility issue (in CDCl3, CD3CN CD3OD) and very less monomer NMR was not recorded in DMSO. The compound was characterized in LCMS. [M+Na]+ calcd for C28H27N3O3SNa was 508.5918, found 508.2. [00665] 5′-S-morpholino cytidine monomer (S-113) was synthesized according to Scheme 13
Figure imgf000205_0004
- -
A/,N-dimethyl- phosphoramic dichloride DBU, LiBr
DCM:ACN (1 :1)
Figure imgf000205_0002
Figure imgf000205_0001
S-112 S-113
Scheme 13
Figure imgf000205_0003
dihydroxytetrahydrothiophen-2-yl)pyrimidin-2(lH)-one (S-108): To a clear solution of (lS)-4- azanyl-l-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl]pyrimi din-2- one (1 g, 3.86 mmol) in anhydrous DMF (10 mL) were added imidazole (656.43 mg, 9.64 mmol) and TBDMSCI (726.63 mg, 4.82 mmol) . The reaction mixture was allowed to stir at rt for 6 hr. Solvent was removed in reduced pressure to get the crude solid residue which was triturated with 70 % EtOAc-Hexane five times to afford (lS)-4-azanyl-l-[(2R,3R,4S,5R)-5-[[l,l- dimethylethyl(dimethyl)silyl]oxymethyl]-3,4-dihydroxy-tetrahydrothiophen-2-yl]pyrimidin-2-one (1.28 g, 3.43 mmol, 88.85% yield) as gummy solid which was used for the next step without any further purification. The compound was characterized in LCMS. [M+H]+ calcd for C15H28N3O4SS1 was 373.54, found 374.1.
Figure imgf000206_0001
S-109
[00667] 4-atnino-l-((2R, 6S)-6-(( (tert-butyldimethylsilyl)oxy)methyl)thiomorpholin-2- yl)pyrinddin-2(lH)-one (S-109): To a suspension of (lS)-4-azanyl-l-[(2R,3R,4S,5R)-5-[[l,l- dimethylethyl(dimethyl)silyl]oxymethyl]-3,4-dihydroxy-tetrahydrothiophen-2-yl]pyrimidin-2-one (1 g, 2.68 mmol) in 80 % MeOH-DCM (60 mL) were added NalCh (715.75 mg, 3.35 mmol) and (NH4)2B4O?.4H2O (2.19 g, 8.32 mmol) and the reaction mixture was allowed to stir at rt for 1.5 hr. Then the solvent was filtered through celite pad and to the filtrate were added MS (4 A0) , NaBHsCN (336.46 mg, 5.35 mmol) and AcOH (160.76 mg, 2.68 mmol, 153.11 pL) . The resulting reaction mixture was allowed to stir for another 2 hr. The reaction mixture was filtered through celite pad and the filtrate was concentrated in reduced pressure to get the crude residue which was triturated with 50 % EtOAC -Hexane five times to afford (lS)-4-azanyl-l-[(2R,6S)-6- [[1,1 -dimethylethyl(dimethyl)silyl]oxymethyl]thiomorpholin-2-yl]pyrimidin-2-one (105 mg, 294.48 pmol, 11.00% yield) as gummy solid which was used for the next step without any further purification. The compound was characterized in LCMS. [M+H]+ calcd for C15H29N4O2SS1 was 357.57, found 358.4.
Figure imgf000206_0002
S-110
[00668] 4-anuno-l-((2R, 6S)-6-(( (tert-butyldimethylsilyl)oxy)methyl)-4-tritylthiomorpholin-
2-yl)pyrinddin-2(lH)-one(S-110): To a suspension of (lS)-4-azanyl-l-[(2R,6S)-6-[[l,l- dimethylethyl(dimethyl)silyl]oxymethyl]thiomorpholin-2-yl]pyrimidin-2-one (81 mg, 227.17 pmol) in anhydrous DMF (3 mL) were added EtsN (68.96 mg, 681.52 pmol, 94.99 pL) and Ph3CCl (69.66 mg, 249.89 pmol) . The reaction mixture was allowed to stir at rt for 2 hr. Then the reaction mixture was diluted with EtOAc and washed with water 5 times to remove DMF followed by brine wash. The organic layer was dried over Na2SO4, fdtered and concentrated to get the crude product which was purified by silica gel column chromatography eluting with 0-8 % MeOH-DCM to afford (lS)-4-azanyl-l-[(2R,4S,6S)-6-[[l,l- dimethylethyl(dimethyl)silyl]oxymethyl]-4-(triphenylmethyl)thiomorpholin-2-yl]pyrimidin-2-one (120 mg, 200.38 pmol, 88.20% yield) as white solid. The compound was characterized in LCMS. [M+H]+ calcd for C34H43N4O2SSi was 598.88, found 598.2.
[00669] 5'-S-morpholino adenosine monomer (S-120) was synthesized according to Scheme
Figure imgf000207_0001
S-120
[00670] ((2S,6R)-6-(6-benzanudo-9H-purin-9-yl)-4-tritylthiomorpholin-2-yl)methyl dimethylphosphoranudochloridate (S-120): N-6-benzoyl protection of compound S-114 is done using BzCl in presence of TMSC1 and pyridine in DCM. TBDMS protection of 5'-OH of compound S-115 is done with TBDMSCI and imidazole in DMF. NalCh mediated diol cleavage and reductive amination of S-116 is done using (NH4)2B?O7.4H2O, NaBFECN, MS (4 A°) and AcOH in 80 % MeOH-DCM to have 5'-S-morpholino adenosine S-117. Morpholino NH is protected with PhsCCI in presence of Et?N in DMF to have S-118. TBAF (IM in THF) mediated TBDMS removal of S- 118 is done to have 6'-OH containing S-119. Chlorophosphoramidate activation of 6'-OH of S-119 is done using A. A-dimethylphosphoramic dichloride in presence of DBU, LiBr in (1 : 1) DCM-ACN to have activated 5'-thiomorpholino adenosine monomer S-120.
[00671] 5'-S-morpholino guanosine monomer (S-127) was synthesized according to Scheme
15
Figure imgf000208_0001
S-127
[00672] ((2S,6R)-6-(6-chloro-2-isobutyramido-9H-purin-9-yl)-4-tritylthiomorpholin-2- yl)methyl dimethylphosphoramidochloridate (S-127): N-2-isobutyric protection of compound S-
121 is done using isobutyric anhydride in presence of HMDS in pyridine to have isobutyric protected guanosine S-122. TBDMS protection of 5'-OH of compound S-122 is done with TBDMSCI and imidazole in DMF to have S-123. NalCU mediated diol cleavage and reductive amination of S-123 is done using (NH4)2B?O7.4H2O, NaBHsCN, MS (4 A°) and AcOH in 80 % MeOH-DCM to have 5'-S-morpholino guanosine S-124. Morpholino NH is protected with PhsCCl in presence of EtsN in DMF to have S-125. TBAF (IM in THF) mediated TBDMS removal of S- 125 is done to have 6'-OH containing S-126. Chlorophosphoramidate activation of 6'-OH of S-126 is done using A. A-dimethylphosphoramic dichloride in presence of DBU, LiBr in (1 : 1) DCM-ACN to have activated 5'-thiomorpholino-6-chloro guanosine monomer S-127. Example 4: Synthesis of A- methyl piperazino (chlorophosphoramidates) monomers
[00673] A-methyl piperazino (chlorophosphoramidates) monomers were synthesized according to Scheme 16.
Figure imgf000209_0001
yl)methyl dimethylphosphoramidochloridate (N-227): TBDPS protection of 5'-OH of compound N-2071,2 is done using TBDPSC1 in presence of imidazole in DMF to have N-211. NaKh mediated diol cleavage and reductive amination using (NH4)2B?O7.4H2O, NaBFFCN, MS (4 A°) and AcOH in 80 % MeOH-DCM to have A-acetyl piperazino uridine N-215. Trityl protection of 3'-NH is done with trityl chloride in presence of triethylamine in DMF to have N-219. 6'-OTBDPS removal using IM TBAF (in THF) and activation of 6'-free OH (N-223) is done using N,N-dimethyl phosphoramic dichloride in presence of DBU and LiBr in (1:1) DCM-ACN to have N-227.
Figure imgf000210_0001
tritylpiperazin-2-yl)methyl dimethylphosphoramidochloridate (N-228): TBDPS protection of 5'- OH of compound N-2081,2 is done using TBDPSC1 in presence of imidazole in DMF to have N- 212. NaIO4 mediated diol cleavage and reductive amination using (NFLCBvOvAHiO, NaBHsCN, MS (4 A°) and AcOH in 80 % MeOH-DCM to have A-acetylpiperazino cytosine N-216. Trityl protection of 3'-NH is done with trityl chloride in presence of tri ethylamine in DMF to have N- 220. 6'-OTBDPS removal using IM TBAF (in THF) and activation of 6'-free OH (N-224) is done using N,N-dimethyl phosphoramic dichloride in presence of DBU and LiBr in (1 : 1) DCM-ACN to have N-228.
Figure imgf000210_0002
N-229
[00676] ((2S,6R)-l-acetyl-6-(6-benzanudo-9H-purin-9-yl)-4-tritylpiperazin-2-yl)tnethyl dimethylphosphoranudochloridate (N-229): TBDPS protection of 5'-OH of compound N-2091,2is done using TBDPSC1 in presence of imidazole in DMF to have N-213. NaIO4 mediated diol cleavage and reductive amination using (NH4)2B?O7.4H2O, NaBHsCN, MS (4 A°) and AcOH in 80 % MeOH-DCM to have A-acetylpiperazino adenosine N-217. Trityl protection of 3'-NH is done with trityl chloride in presence of triethylamine in DMF to have N-221. 6'-OTBDPS removal using IM TBAF (in THF) and activation of 6'-free OH (N-225) is done using N,N-dimethyl phosphoramic dichloride in presence of DBU and LiBr in (1:1) DCM-ACN to have N-229.
Figure imgf000210_0003
N-230 [00677] ((2S,6R)-l-acetyl-6-(2-isobutyramido-6-oxo-9H-6l5-purin-9-yl)-4-tritylpiperazin-2- yl)methyl dimethylphosphoranudochloridate (N-230): TBDPS protection of 5'-OH of compound N-2101,2is done using TBDPSC1 in presence of imidazole in DMF to have N-214. NalCh mediated diol cleavage and reductive amination using (NH4)2B?O7.4H2O, NaBHsCN, MS (4 A°) and AcOH in 80 % MeOH-DCM to have A-acetylpiperazino guanosine N-218. Trityl protection of 3'-NH is done with trityl chloride in presence of triethylamine in DMF to have N-222. 6'-OTBDPS removal using IM TBAF (in THF) and activation of 6'-free OH (N-226) is done using N,N-dimethyl phosphoramic dichloride in presence of DBU and LiBr in (1:1) DCM-ACN to have N-230.
References:
1. Hernandez, D.; Boto, A. Nucleoside analogues: synthesis and biological properties of azanucleoside derivatives. Eur. J. Org. Chem. 2014, 2014 (11), 2201-2220.
2. Reist, E. J.; Gueffroy, D. E.; Blackford, R. W.; Goodman, L. Pyrrolidine Sugars. Synthesis of 4'-Acetamidoadenosine and Other Derivatives of 4-Amino-4-deoxy-D- nbosel. J. Org. Chem. 1966, 31 (12), 4025-4030.
[00678] Abbreviations used in nucleotide sequences described herein are summarized in Table
7 and in FIG. 14.
Table 7: Abbreviations used in sequences
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
[00637] All patents and other publications identified in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[00638] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. Further, to the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated can be further modified to incorporate features shown in any of the other embodiments disclosed herein.

Claims

CLAIMS What is claimed is
1. A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein each strand is independently 15 to 35 nucleotides in length, and wherein the first or second strand comprises at least one nucleoside of Formula (IV):
Figure imgf000225_0001
Formula (IV) wherein:
B’ is an optionally substituted nucleobase;
XM is CH2, O, NRN or S, where RN 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;
R43 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 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2- soalkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxy alkyl (e.g., 2- methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or a nitrogen protecting group;
R45 represents a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker covalently bonded 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, alkoxy alkyl (e.g., 2-methoxy ethyl), alkoxy alkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (H0)(NH2)(0)P-0- 5'), alkylphosphonates [(Rp)(0H)(0)P-0-5', Rp is optionally substituted C1-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(Rpl)(OH)(O)P-O- 5', RP1 is alkoxyalkyl, e.g., methoxymethyl (CFPOMe) or ethoxymethyl ], (H0)2(X)P- O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O- P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[-(CH2)a-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group (e.g., -CH =CH-XP, Xp is a phosphonate group) or C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), wherein
X is O or S; a and b are each independently 1-10, provided that only one of R43 and R45 is a solid support or linker covalently bonded to a solid support; and each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl, and provided that,
(i) when R43 is not a bond to an intemucleotide linkage to a subsequent nucleotide, then
R45 is a bond to an intemucleotide linkage to a preceding nucleotide; and
(ii) when R45 is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R43 is a bond to an intemucleotide linkage to a preceding nucleotide.
2. The double-stranded nucleic acid of claim 1, wherein XM is CH2.
3. The double-stranded nucleic acid of claim 1, wherein XM is O.
4. The double-stranded nucleic acid of claim 1, wherein XM is S.
5. The double-stranded nucleic acid of any one of claims 1-4, wherein R43 is bond to an intemucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, a nitrogen protecting group, a linker, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, or a linker covalently bonded to one or more lipids.
6. The double-stranded nucleic acid of claim 6, wherein R43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
7. The double-stranded nucleic acid of claim 6, wherein R43 is either (i) hydrogen or a nitrogen protecting group; or (ii) hydroxyl or a protected hydroxyl.
8. The double-stranded nucleic acid of any one of claims 1-7, wherein R45 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 R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
9. The double-stranded nucleic acid of claim 8, wherein R45 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 R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
10. The double-stranded nucleic acid of claim 9, wherein R45 is a bond to an intemucleotide linkage to a preceding nucleotide.
11. The double-stranded nucleic acid of claim 9, wherein R45 is optionally substituted C1-30 alkoxy or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
12. The double-stranded nucleic acid of claim 9, wherein R45 is hydroxyl.
13. The double-stranded nucleic acid of any one of claims 1-12, 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.
14. The double-stranded nucleic acid of any one of claims 1-13, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one ribonucleotide.
15. The double-stranded nucleic acid of any one of claims 1-14, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one 2’-deoxyribonucleotide.
16. The double-stranded nucleic acid of any one of claims 1-15, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a modified or non-natural nucleobase in addition to the nucleotide of Formula (IV).
17. The double-stranded nucleic acid of any one of claims 1-16, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a modified ribose sugar in addition to the nucleotide of Formula (IV).
18. The double-stranded nucleic acid of any one of claims 1-17, wherein the strand comprising the nucleotide of Formula (IV) 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).
19. The double-stranded nucleic acid of any one of claims 1-18, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a 2’-F ribose in addition to the nucleotide of Formula (IV).
20. The double-stranded nucleic acid of any one of claims 1-19, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a 2’- OMe ribose in addition to the nucleotide of Formula (IV).
21. The double-stranded nucleic acid of any one of claims 1-20, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide comprising a moiety other than a ribose sugar in addition to the nucleotide of Formula (IV).
22. The double-stranded nucleic acid of any one of claims 1-21, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one modified intemucleotide linkage.
23. The double-stranded nucleic acid of any one of claims 1-22, wherein at least one of the first or second strand comprises at least one ligand.
24. The double-stranded nucleic acid of claim 23, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one ligand.
25. The double-stranded nucleic acid of any one of claims 1-24, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the strand comprising the nucleotide of Formula (IV).
26. The oligonucleotide of any one of claims 1-25, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the strand comprising the nucleotide of Formula (IV)., and wherein the nucleoside of Formula (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphodiester intemucleotide linkage.
27. The double-stranded nucleic acid of any one of claims 1-26, wherein the nucleoside of Formula (IV) is present at 3’-end of the strand comprising the nucleotide of Formula (IV)., and wherein the nucleoside of Formula (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphorothioate internucleotide linkage.
28. The double-stranded nucleic acid of any one of claims 1-27, wherein the first and second strand are independently 18 to 25 nucleotides in length.
29. The double-stranded nucleic acid of any one of claims 1-28, wherein the first and second strand form a double-stranded or duplex region of 17 to 25 basepairs.
30. The double-stranded nucleic acid any one of claims 1-29, wherein double-stranded nucleic acid is capable of inducing RNA interference.
31. The double-stranded nucleic acid of any one of claims 1-30, wherein one or both strands have a 1 – 5 nucleotide overhang on its respective 5’-end or 3’-end.
32. The double-stranded nucleic acid of any one of claims 1-31, wherein only one strand has a 2 nucleotide overhang on its 5’-end or 3’-end.
33. The double-stranded nucleic acid of any one of claims 1-32, wherein only one strand has a 2 nucleotide overhand on its 3’-end.
34. An oligonucleotide comprising at least one nucleoside of Formula (IV):
Figure imgf000229_0001
Formula (IV) , wherein: B’ is an optionally substituted nucleobase; XM is CH2, O, NRN or S, where RN 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; R43 is a bond to an internucleotide 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 C2-30alkenyl, optionally substituted C2- 30alkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2- methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, -0-C4-3oalkyl-
ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or a nitrogen protecting group;
R45 represents a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker covalently bonded 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, alkoxy alkyl (e.g., 2-methoxy ethyl), alkoxy alkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (H0)(NH2)(0)P-0- 5'), alkylphosphonates [(Rp)(0H)(0)P-0-5', Rp is optionally substituted C1-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(Rpl)(OH)(O)P-O- 5', RP1 is alkoxyalkyl, e.g., methoxymethyl (CFPOMe) or ethoxymethyl ], (H0)2(X)P- O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O- P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[-(CH2)a-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group (e.g., -CH=CH-XP, Xp is a phosphonate group), C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), wherein
X is O or S; a and b are each independently 1-10, provided that only one of R43 and R45 is a solid support or linker covalently bonded to a solid support; and each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl, and provided that, (i) when R43 is not a bond to an intemucleotide linkage to a subsequent nucleotide, then
R45 is a bond to an intemucleotide linkage to a preceding nucleotide; and
(ii) when R45 is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R43 is a bond to an intemucleotide linkage to a preceding nucleotide.
35. The oligonucleotide of claim 34, wherein R43 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.
36. The oligonucleotide of claim 35, wherein R43 is a bond to an intemucleotide linkage to a subsequent nucleotide.
37. The oligonucleotide of claim 35, wherein R43 is a solid support, or a linker (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) covalently bonded to a solid support, hydrogen or a nitrogen protecting group.
38. The oligonucleotide of any one of claims 34-37, wherein R45 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 R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
39. The oligonucleotide of claim 38, wherein R45 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 R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group..
40. The oligonucleotide of claim 39, wherein R45 is a bond to an intemucleotide linkage to a preceding nucleotide.
41. The oligonucleotide of claim 39, wherein R45 is hydroxyl, protected hydroxyl, or optionally substituted C1-30 alkoxy, or R45 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.
42. The oligonucleotide of any one of claims 34-41, wherein XM is CH2.
43. The oligonucleotide of any one of claims 34-41, wherein XM is O.
44. The oligonucleotide of any one of claims 34-41, wherein XM is S.
45. The oligonucleotide of any one of claims 34-44, 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.
46. The oligonucleotide of any one of claims 34-45, wherein the oligonucleotide comprises from 3 to 50 nucleotides.
47. The oligonucleotide of any one of claims 34-46, wherein the oligonucleotide comprises at least one ribonucleotide.
48. The oligonucleotide of any one of claims 34-47, wherein the oligonucleotide comprises at least one 2’-deoxyribonucleotide.
49. The oligonucleotide of any one of claims 34-48, wherein the oligonucleotide comprises at least one nucleotide with a modified or non-natural nucleobase in addition to the nucleotide of Formula (IV).
50. The oligonucleotide of any one of claims 34-49, wherein the oligonucleotide comprises at least one nucleotide with a modified ribose sugar in addition to the nucleotide of Formula (IV).
51. The oligonucleotide of any one of claims 34-50, 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).
52. The oligonucleotide of any one of claims 34-51, wherein the oligonucleotide comprises at least one nucleotide with a 2’-F ribose in addition to the nucleotide of Formula (IV).
53. The oligonucleotide of any one of claims 34-52, wherein the oligonucleotide comprises at least one nucleotide with a 2’-0Me ribose in addition to the nucleotide of Formula (IV).
54. The oligonucleotide of any one of claims 34-53, wherein the oligonucleotide comprises at least one nucleotide comprising a moiety other than a ribose sugar in addition to the nucleotide of Formula (IV).
55. The oligonucleotide of any one of claims 34-54, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.
56. The oligonucleotide of any one of claims 34-55, wherein the oligonucleotide is attached to a solid support.
57. The oligonucleotide of any one of claims 34-56, wherein oligonucleotide comprises at least one ligand.
58. The oligonucleotide of any one of claims 34-57, wherein the oligonucleotide comprises at least one hydroxyl, phosphate or amino protecting group.
59. The oligonucleotide of any one of claims 34-58, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide.
60. The oligonucleotide of any one of claims 34-59, 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.
61. The oligonucleotide of any one of claims 34-60, 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.
62. 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 claims 1-33 or 92-120, wherein the first strand or the second strand is complementary to a target gene; or
(ii) an oligonucleotide according to any one of claims 34-61 or 86-91, wherein the oligonucleotide is complementary to a target gene.
63. A compound of Formula (III):
Figure imgf000233_0001
Formula (III) wherein:
B’ is an optionally substituted nucleobase;
XM is CH2, O, NRN or S, where RN 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;
R33 is hydrogen, hydroxy, protected hydroxy, 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 C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R8)(CH2R9), or -O-C4-30alkyl- ON(CH2R8)(CH2R9); R35 is hydroxy, protected hydroxy, phosphate group, a reactive phosphorous group, optionally substituted C1-30 alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R8)(CH2R9), -O-C4-30alkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH- 5', (HO)(NH2)(O)P-O-5'), alkylphosphonates (R(OH)(O)P-O-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc…), alkyletherphosphonates (R(OH)(O)P-O-5', R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc…), (HO)2(X)P-O[- (CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[-(CH2)a-O- P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a- P(X)(OH)-O]b- 5', H[-(CH2)a-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', wherein X is O or S; and a and b are each independently 1-10), provided that only one of R33 and R35 is a reactive phosphorous group; or R35 taken together with the carbon to which it is attached form a vinylphosphonate (VP) group or C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate), a vinylphosphonate (VP) group; and each R8 and R9 is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30alkynyl.
64. The compound of claim 63, wherein R33 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.
65. The compound of claim 63, wherein R33 is (i) a H or nitrogen protecting group or (ii) hydroxy or protected hydroxy.
66. The compound of any one of claims 63-65, wherein R35 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 R35 taken together with the carbon to which it is attached form a vinylphosphonate group.
67. The compound of any one of claims 63-66, wherein R35 is a reactive phosphorous group, linker, solid support, a linker covalently bonded to a solid support, hydroxyl, or a protected hydroxyl.
68. The compound of any one of claims 63-67, wherein R35 is reactive phosphorous group, solid support, or a linker covalently bonded to a solid support.
69. The compound of any one of claims 63-68, wherein R35 is -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 is halogen (e.g., Cl)
70. The compound of any one of claims 63-69, wherein XM is CH2.
71. The compound of any one of claims 63-69, wherein XM is O.
72. The compound of any one of claims 63-69, wherein XM is S.
73. The compound of claim 63, wherein:
XM is CH2;
R33 is H or nitrogen protecting group (e.g., trityl); and
R35 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 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.
74. The compound of claim 63, wherein:
XM is O;
R33 is H or nitrogen protecting group (e.g., trityl); and R35 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 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.
75. The compound of claim 63, wherein:
XM is S;
R33 is H or nitrogen protecting group (e.g., trityl); and
R35 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 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.
76. The compound of claim 63, wherein:
XM is CH2;
R35 is hydroxyl or a protected hydroxyl; and
R33 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.
77. The compound of claim 76, wherein:
XM is CH2;
R35 is hydroxyl or a protected hydroxyl; and
R33 is a solid support or a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support.
78. The compound of claim 63, wherein:
XM is O;
R35 is hydroxyl or protected hydroxyl; and
R33 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.
79. The compound of claim 78, wherein:
XM is O;
R35 is hydroxyl or protected hydroxyl; and
R33 is a solid support, or a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support.
80. The compound of claim 63, wherein:
XM is S;
R35 is hydroxyl or protected hydroxyl; and
R33 is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XD is O or S; each RP2 is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4 is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.
81. The compound of claim 80, wherein:
XM is S;
R35 is hydroxyl or protected hydroxyl; and
R33 is a solid support, or a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support.
82. The compound of any one of claims 63-81, 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.
83. The compound of any one of claims 63-82, wherein B and B’ are independently adenine, cytosine, guanine, thymine, uracil,
Figure imgf000237_0001
Figure imgf000237_0002
Figure imgf000238_0001
selected from 1 to 10; and R1 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.
A compound selected from the group consisting of:
Figure imgf000239_0001
Figure imgf000240_0001
(where @ is a solid support, e.g., CPG),
Figure imgf000240_0002
Figure imgf000240_0003
(where X is O, CH2. NR or S, and R is a protecting group or an
Figure imgf000241_0001
85. An oligonucleotide prepared using a compound of any one of claims 63-84.
86. The oligonucleotide of any one of claims 34-61, wherein the nucleotide of Formula (IV) is at one of positions 2-9, counting from the 5 ’end of the oligonucleotide.
87. The oligonucleotide of claim 86, 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.
88. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 5, counting from the 5 ’end of the oligonucleotide.
89. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 6, counting from the 5 ’end of the oligonucleotide.
90. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 7, counting from the 5 ’end of the oligonucleotide.
91. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 8, counting from the 5 ’end of the oligonucleotide.
92. 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 of any one of claims 34-61 or 86- 91.
93. The double-stranded nucleic acid of claim 92, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the sense strand.
94. The double-stranded nucleic acid of claim 93, 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).
95. The double-stranded nucleic acid of claim 94, 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).
96. The double-stranded nucleic acid of claim 94, 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).
97. The double-stranded nucleic acid of claim 94, 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).
98. The double-stranded nucleic acid of claim 94, 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).
99. The double-stranded nucleic acid of claim 94, 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).
100. The double-stranded nucleic acid of claim 92, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the antisense strand.
101. The double-stranded nucleic acid of claim 100, 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).
102. The double-stranded nucleic acid of claim 101, 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).
103. The double-stranded nucleic acid of claim 101, 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.
104. The double-stranded nucleic acid of claim 101, 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.
105. The double-stranded nucleic acid of claim 101, 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.
106. The double-stranded nucleic acid of claim 101, 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 nucleotidefrom the 5 ’-end of the antisense strand.
107. The double-stranded nucleic acid of any one of claims 1-33, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the sense strand.
108. The double-stranded nucleic acid of claim 107, 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).
109. The double-stranded nucleic acid of claim 108, 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).
110. The double-stranded nucleic acid of claim 107, 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).
111. The double-stranded nucleic acid of claim 107, 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).
112. The double-stranded nucleic acid of claim 107, 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).
113. The double-stranded nucleic acid of claim 107, 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).
114. The double-stranded nucleic acid of any one of claims 1-33, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the antisense strand.
115. The double-stranded nucleic acid of claim 114, 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).
116. The double-stranded nucleic acid of claim 115, 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).
117. The double-stranded nucleic acid of claim 115, 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.
118. The double-stranded nucleic acid of claim 115, 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.
119. The double-stranded nucleic acid of claim 115, 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.
120. The double-stranded nucleic acid of claim 115, 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.
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