EP4709855A2 - Oligonucléotides à boucle simple brin - Google Patents

Oligonucléotides à boucle simple brin

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Publication number
EP4709855A2
EP4709855A2 EP24731742.3A EP24731742A EP4709855A2 EP 4709855 A2 EP4709855 A2 EP 4709855A2 EP 24731742 A EP24731742 A EP 24731742A EP 4709855 A2 EP4709855 A2 EP 4709855A2
Authority
EP
European Patent Office
Prior art keywords
nucleotides
stranded oligonucleotide
nucleotide
formula
modified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP24731742.3A
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German (de)
English (en)
Inventor
Muthiah Manoharan
Ivan Zlatev
Dhrubajyoti Datta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Alnylam Pharmaceuticals Inc
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Alnylam Pharmaceuticals Inc
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Publication date
Application filed by Alnylam Pharmaceuticals Inc filed Critical Alnylam Pharmaceuticals Inc
Publication of EP4709855A2 publication Critical patent/EP4709855A2/fr
Pending legal-status Critical Current

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • This invention generally relates to the field of RNA interference technology with single- stranded loop oligonucleotides.
  • RNAi agents Chemical modifications of the nucleobases, ribose sugar, and phosphate backbone have been used in double-stranded RNAi agents to improve drug-like properties of these therapeutic oligonucleotides and to confer favorable pharmacological properties to GalNAc- oligonucleotide conjugates in preclinical and clinical development.
  • siRNA designs have been developed to achieve better stability and potency.
  • the current studies addressed the stability and duration-related challenges by incorporating chemical modifications, but overlooked process-related challenges in synthesizing the double-stranded siRNAs.
  • Sense and antisense strands are typically synthesized separately, go through a tedious multistep purification as single strands, and then annealed into a duplex which further undergoes another round of purification and quality control. This process is complex, time-taking, expensive, and raises environmental sustainability concerns.
  • One aspect of the invention relates to a single-stranded oligonucleotide capable of inhibiting the expression of a target gene, having a sequence represented by formula (I):
  • Z 1 is a first oligonucleotide, comprising 10-100 optionally modified nucleotides (e.g., 15-100) that is substantially complementary to a target gene;
  • Z 2 is a second oligonucleotide, comprising 10-100 optionally modified nucleotides (e.g., 15-100) that is substantially complementary to Z 1 ;
  • Z 1 and Z 2 are capable of forming an intra-strand duplexed region comprising 3 or more consecutive base pairs;
  • L is a linking group
  • Q 1 and Q 2 each independently represent 0 to 12 optionally modified nucleotides; and at least one nucleotide in formula (I) is a modified nucleotide.
  • the first oligonucleotide Z 1 and second oligonucleotide Z 2 each may independently comprise 15 - 100 optionally modified nucleotides.
  • Z 1 and Z 2 each may independently comprise 15 - 40, 15 - 25, or 19 - 23 optionally modified nucleotides.
  • the first oligonucleotide Z 1 and second oligonucleotide Z 2 each may independently comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • Z 1 and Z 2 each may independently have about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 15 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, about 10 to about 20 nucleotides, about 15 to about 50 nucleotides, about 15 to about 40 nucleotides, about 15 to about 35 nucleotides, about 15 to about 30 nucleotides, about 15 to about 25 nucleotides, about 15 to about 20 nucleotides, about 19 to about 23 nucleotides, about 19 to about 21 nucleotides, or about 18 to about 20 nucleotides in length.
  • Each of the nucleotides in first oligonucleotide Z 1 and second oligonucleotide Z 2 may be independently and optionally modified. In some embodiments, Z 1 and Z 2 each contain the same number of optionally modified nucleotides.
  • Q 1 and Q 2 each may independently comprise 0 to 12 optionally modified nucleotides.
  • Q 1 and Q 2 each may independently comprise 0 to 10, 0 to 6, 0 to 4, 0 to 3, 0 to 2, 1 to 6, 1 to 4, 1 to 3, or 2 to 3 optionally modified nucleotides.
  • Q 1 and Q 2 each are 0.
  • one of Q 1 and Q 2 is 0.
  • Q 1 and Q 2 have the same number of optionally modified nucleotides.
  • the single-stranded oligonucleotide can be cleaved at the linking group L.
  • the first oligonucleotide Z 1 can be cleaved into an antisense strand that is substantially complementary to a target gene (e.g., a target mRNA or DNA), and the second oligonucleotide Z 2 can be cleaved into a sense strand that is substantially complementary to Z 1 .
  • the first oligonucleotide Z 1 and second oligonucleotide Z 2 can form an intramolecular double-stranded region comprising 3 or more consecutive base pairs (e.g., a duplex region of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 base pairs).
  • the duplex region may comprise 10-25, 15-25, 19-23, 19, 20, 21, 22, or 23 base pairs.
  • the intra-strand duplexed region formed by Z 1 and Z 2 may contain all consecutive base pairs, or may contain no more than 3 (e.g., 0, 1, 2, or 3) mismatch based pairs.
  • the single-stranded oligonucleotide comprises at least one chemical modification.
  • each of the first oligonucleotide Z 1 and second oligonucleotide Z 2 comprise at least one chemical modification.
  • all the nucleotides in Z 2 are modified nucleotides.
  • all the nucleotides in Z 1 are modified nucleotides.
  • all the nucleotides of the single-stranded oligonucleotide are modified.
  • the chemical modification to the nucleotide(s) may include an intemucleoside linkage modification, a nucleobase modification, a sugar modification, or combinations thereof.
  • the chemical modification is selected from the group consisting of LNA, ENA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'- O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-alkyl (e.g., 2'-OMethyl), 2'-O-allyl, 2'- C- allyl, 2' -fluoro, 2' -deoxy, 2'-O-N-methylacetamido ( 2'-O-NMA), 2'-O- dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L- nucleoside modification (such as 2'-modified L-nucleoside, e.g., 2'-deoxy-L-nucleo
  • the chemical modification is a 2' -modification selected from the group consisting of 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O- methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, 2'-fluoro, and combinations thereof.
  • about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 1 are modified.
  • about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 2 are modified.
  • about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of all the nucleotides in the single-stranded oligonucleotide are modified. For example, when 50% of all the nucleotides are modified, 50% of all nucleotides present in the single-stranded oligonucleotide contain at least one modification as described herein.
  • At least 50% of the nucleotides of the single-stranded oligonucleotide are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, or 2' -fluoro.
  • one or more of the five internucleotide linkages among the six 3'-terminal nucleotides is a modified intemucleotide linkage. In some embodiments, one or more of the five intemucleotide linkages among the six 5'-terminal nucleotides is a modified intemucleotide linkage.
  • one or more of the five intemucleotide linkages among the six 5'-terminal nucleotides of Z 2 is a modified intemucleotide linkage. In some embodiments, one or more of the five intemucleotide linkages among the six 5'-terminal nucleotides of Z 1 is a modified intemucleotide linkage.
  • the single-stranded oligonucleotide further comprises one or more modified intemucleotide linkage between the 3'-terminal nucleotide of Z 1 and the first nucleotide of Q 1 . In some embodiments, the single-stranded oligonucleotide further comprises one or more modified intemucleotide linkages between the nucleotides of Q 1 . [0022] In some embodiments, the single-stranded oligonucleotide further comprises a phosphate or phosphate mimic at the 5'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the single-stranded oligonucleotide comprises a phosphate mimic at the 5'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ). In one embodiment, at least one phosphate mimic is at the 5' end of Z 1 . In one embodiment, the phosphate mimic is a 5'- vinyl phosphonate (VP). In one embodiment, the phosphate mimic is a 5' -cyclopropyl phosphonate. In one embodiment, the phosphate mimic is a 5'-vinyl phosphate.
  • VP vinyl phosphonate
  • the phosphate mimic is a 5'-cyclopropyl phosphonate.
  • the 5' -end or 3' -end nucleotide in the single-stranded oligonucleotide of formula (I) comprise a 2'-5'-linked nucleotide modification; or the 5'-end or 3' -end nucleotide is conjugated to an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide (e.g., ribonucleotide), optionally via a phosphodiester, phosphorothioate, or phosphodithioate linkage.
  • the 5' -end or 3' -end nucleotide in the single-stranded oligonucleotide of formula (I) is modified to comprise a linking moiety containing a mono-, di-, tri-, tetra-, penta- or polyprolinol, or mono-, di-, tri-, tetra-, penta- or polyhy droxyprolinol .
  • the single-stranded oligonucleotide further comprises at least one terminal, chiral modification (such as a terminal, chiral phosphorus atom).
  • a site specific, chiral modification to the intemucleotide linkage may occur at the 5' end, 3' end, or both the 5' end and 3' end of a nucleotide sequence. This is being referred to herein as a “terminal, chiral'' modification.
  • the terminal modification may occur at a 3' or 5' terminal position in a terminal region, e.g., at a position on a terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a nucleotide sequence.
  • Each of the chiral pure phosphorus atoms may be in either Rp configuration or Sp configuration, and combination thereof. More details regarding chiral modifications and chirally-modified RNA agents can be found in WO 2019/126651 Al, which is incorporated herein by reference in its entirety.
  • the single-stranded oligonucleotide comprises at least two consecutive phosphorothioate or methylphosphonate intemucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least two blocks of two consecutive phosphorothioate or methylphosphonate internucleotide linkage modifications. In some embodiments, the single-stranded oligonucleotide comprises at least three blocks of two consecutive phosphorothioate or methylphosphonate internucleotide linkage modifications.
  • the single-stranded oligonucleotide has at least two phosphorothioate internucleotide linkages at the first five nucleotides on a nucleotide sequence (counting from the 5' end) (e.g., Z 1 and/or Z 2 ).
  • a nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 1 and/or Z 2 ) comprises two blocks of one, two, or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.
  • a nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 1 and/or Z 2 ) comprises at least two consecutive phosphorothioate intemucleotide linkage modifications within positions 18-23 of the nucleotide sequence, counting from the 5' -end of the nucleotide sequence.
  • a nucleotide sequence of the single- stranded oligonucleotide (e.g., Z 1 and/or Z 2 ) comprises at least two consecutive phosphorothioate intemucleotide linkage modifications within position 1-5 of the nucleotide sequence, counting from the 5'-end of the nucleotide sequence.
  • each of Z 1 and Z 2 of the single-stranded oligonucleotide comprises at least two consecutive phosphorothioate intemucleotide linkage modifications.
  • each of Z 1 and Z 2 of the single-stranded oligonucleotide comprises: at least two consecutive phosphorothioate intemucleotide linkage modifications within positions 18-23 of the nucleotide sequence, and at least two consecutive phosphorothioate intemucleotide linkage modifications within position 1-5 of the nucleotide sequence, counting from the 5 '-end of the nucleotide sequence.
  • the target gene may be a mRNA, pre-mRNA, microRNA, pre-miRNA, long non-coding RNA (IncRNA), or DNA.
  • the single-stranded oligonucleotide may be an inhibitory single-stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, microRNA mimic, supermir, aptamer, U1 adaptor, triplex -forming oligonucleotide, RNA activator, immuno-stimulatory oligonucleotide, decoy oligonucleotide, heteroduplex-forming oligonucleotide, or a single-stranded siRNA (ss- siRNA) oligonucleotide.
  • At least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5' -end or the 3' -end of the single-stranded oligonucleotide are not phosphorothioate linkages.
  • the at least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5'-end or the 3' -end of the single-stranded oligonucleotide are each independently a natural phosphate group or a phosphodiester linkage, or a nitrogen-modified phosphorous-containing linkage (PN-linkage).
  • the PN-linkage comprises an optionally substituted cyclic guanidine moiety.
  • the PN-linkage can have the structure of , , wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the PN-linkage is stereochemically controlled.
  • the PN-linkage comprises a triazole moiety (e.g., an optionally substituted triazolyl group).
  • the PN-linkage can have the structure of , wherein W is O or S.
  • W is O.
  • W is S.
  • the PN-linkage is stereochemically controlled.
  • the PN-linkage comprises an alkyne moiety (e.g., an optionally substituted alkynyl group).
  • the PN-linkage can have the structure of wherein W is O or S. In some embodiments, W is
  • W is S.
  • the PN-linkage is stereochemically controlled.
  • the PN-linkage comprises a Tmg group ( ).
  • the PN-linkage can have the structure of , wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S. In some embodiments, the PN-linkage is stereochemically controlled.
  • Additional suitable PN-linkages may include those described in WO 2019/032612 and W02021/030778, which are incorporated herein by reference in their entirety.
  • L of formula (I) is a cleavable linking group.
  • the cleavable linking group is cleavable in a homogenate, tritosome, cytosol, or endosome of any types of cells.
  • the cleavable linking group may be cleavable in liver homogenates, liver tritosomes, liver lysosomes, liver cytosol, liver endosome, brain homogenates, brain tritosomes, brain lysosomes, brain cytosol, or brain endosome.
  • the cleavable linking group is a redox cleavable linker (such as a reductively cleavable linker; e.g., a disulfide group), an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavable linker (e.g., an ester group), a phosphatase cleavable linker (e.g., an ester group), a peptidase cleavable linker (e.g., an ester group), or endosomal cleavable linker (or a protease cleavable linker, e.g., a carbohydrate linker).
  • a redox cleavable linker such as a reductively cleavable linker; e.g., a disulfide group
  • the cleavable linking group is an endosomal cleavable linker or a protease cleavable linker, for instance, a carbohydrate linker, wherein the linker is cleaved at least 1.25 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).
  • L of formula (I) contains a linking moiety represented by a formula: #-(N) n -**.
  • # is the bond to Q 1 and ** is the bond to Q 2 ;
  • n is 3 to 12; and each N is independently a linking moiety.
  • each N may be independently a linking monomer having a chain length of 3 or more atoms.
  • chain length'' refers to the number of atoms in the shortest linear chain formed by the linking monomer. For instance, for a PEG/PEO, having a structure of
  • the chain length of the linking monomer is 3 (triethylene glycol).
  • the chain length of the linking monomer having a formula of is 7. In one embodiment, the chain length of the linking monomer having a formula of
  • one or more linking moieties (N) in L of formula (I) may be an optionally modified nucleotide.
  • one or more linking moieties (N) in L of formula (I) may be independently selected from the group consisting of a 2'-deoxynucleotide (dN), a 2'- deoxy -2' -fluoro nucleotide (fN), a ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'- ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2'-ara ribonucleotide).
  • Ara-nucleotides feature an opposite stereochemistry at the 2' carbon atom compared to ribo-nucleotides.
  • one or more linking moieties (N) in L of formula (I) may contain a modified intemucleotide linkage selected from the group consisting of a phosphodiester, phosphotriester (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), methylenemethylimino, a nitrogen-modified phosphorous-containing linkage (PN-linkage) (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), thiodiester, thionocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, borano phosphate, borano phosphate ester, amide,
  • one or more linking moieties (N) in L of formula (I) may contain a moiety selected from the group consisting of an aliphatic saturated or unsaturated alkyl chain; a phosphorous-containing linkage, including a phosphate, a phosphonate, a phosphoramidate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphodiester, phosphotriester (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), and a nitrogen-modified phosphorous-containing linkage
  • a phosphorous-containing linkage including a phosphate, a phosphonate, a phosphoramidate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphodiester, phosphotriester (optionally comprising the linkage phosphorus atom in either Rp configuration or
  • PN-linkage (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration); a (poly)ethylene glycol chain, including diethylene glycol, triethylene glycol, tetra, penta, hexa, hepta, octa, nona, or deca ethylene glycol; glycerol or glycerol ester; an aminoalkyl ether; and combinations thereof.
  • one or more linking moieties (N) in L of formula (I) may contain a moiety selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.
  • one or more linking moieties (N) in L of formula (I) may be independently selected from the group consisting of
  • one or more linking moieties (N) in L of formula (I) may be independently selected from the group consisting of: wherein:
  • Base is an optionally modified nucleobase
  • R D is a C 4-30 alkyl, C 4-30 alkyenyl, or C 4-30 alkynyl.
  • one or more linking moieties (N) in L of formula (I) comprise a mono-, di-, tri-, tetra-, penta- or poly-prolinol, optionally conjugated with a ligand; a mono-, di-, tri-, tetra-, penta- or poly-hydroxyprolinol, optionally conjugated with a ligand; an optionally modified nucleotide; or combinations thereof.
  • L of formula (I) contains one or more of a mono-, di-, tri-, tetra-, penta- or poly-prolinol, optionally conjugated with a ligand; and one or more optionally modified nucleotides.
  • L of formula (I) contains one or more of a mono-, di-, tri-, tetra-, penta- or poly-hydroxyprolinol, optionally conjugated with a ligand; and one or more optionally modified nucleotides.
  • one or more linking moieties (N) in L of formula (I) comprises a moiety selected from the group consisting of:
  • L of formula (I) contains a linking moiety represented by a formula: #-(N) n -**.
  • # is the bond to Q 1 and ** is the bond to Q 2 ;
  • n is 3 to 12; and each N is independently an optionally modified nucleotide, Y34, Y16, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, QI 1, Q150, Q151, Q173, Q221, Q222, Q367, or Q368.
  • n is 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5. In one embodiment, n is 5.
  • L of formula (I) contains 3-5 of 2'-deoxy nucleotides, a triplet of 2' -deoxy-2' -fluoro nucleotides, a triplet of ribonucleotides, a triplet of 2'-O-methyl nucleotides, or a triplet of Q304.
  • L of formula (I) contains one of the followings:
  • dN represents a 2' -deoxy nucleotide
  • fN represents a 2'-deoxy-2' -fluoro nucleotide
  • rN represents a ribonucleotide
  • mN represents a 2'-O-methyl nucleotide.
  • L of formula (I) contains one of the followings:
  • dN represents a 2' -deoxy nucleotide
  • fN represents a 2'-deoxy-'2 -fluoro nucleotide
  • rN represents a ribonucleotide
  • mN represents a 2'-O-methyl nucleotide.
  • one or more internucleotide linkages between the nucleotides in L may be modified intemucleotide linkages independently selected from the group consisting of a phosphodiester, phosphotriester (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), and a nitrogen-modified phosphorous-containing linkage (PN-linkage) (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration).
  • a phosphodiester optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration
  • phosphotriester optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration
  • hydrogen phosphonate alkyl or
  • L of formula (I) may contain one or more linking moiety selected from the group consisting of a triazole linkage, an amide linkage, a sulfide or disulfide linkage, a phosphate linkage, an oxime linkage, a hydrazo linkage, a N,N'- dialkylenehydrazo linkage, a methyleneimino linkage, a methylenecarbonylamino linkage, a methylenemethylimino linkage, a methylenehydrazo linkage, a methylenedimethylhydrazo linkage, a methyleneoxymethylimino linkage, a hydroxylamino linkage, a formacetal linkage, an alkyl or aryl linkage, a PEG linkage, an ether linkage, a thioether linkage, a thiodiester linkage, a thionocarbamate linkage, a thioacetamido linkage, a
  • L of formula (I) may contain one or more cyclic groups selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] di oxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.
  • L of formula (I) contains a nucleotide-based linker (tether). In some embodiments, L contains a non-nucleotide-based linker (tether).
  • the nucleotide-based or non-nucleotide-based linker (tether) contained in L is a stable linker (tether) that is stable in a biological fluid.
  • the nucleotide-based or non-nucleotide based stable linker (tether) is stable in plasma or artificial cerebrospinal fluid.
  • the cleavable linking group comprises a moiety selected from the group consisting of forumula
  • the cleavable linking group comprises a moiety selected from the following:
  • n 0 or 1-20;
  • n 0 or 1-20; mono-, di-, tri-, tetra-, penta- or polyprolinol, optionally conjugated with a ligand; mono-, di-, tri-, tetra-, penta- or polyhydroxyprolinol, optionally conjugated with a ligand.
  • the cleavable linking group (tether) comprises a nucleic acid linker of 1 to 15 nucleotides in length.
  • the nucleic acid linker may be 2 to 7, 5 to 7, 2 to 5, or 3, 4, or 5 optionally modified nucleotides in length.
  • the cleavable linking group comprises a nucleic acid linker comprising one or more nucleotides selected from the group consisting of 2'-O- methyl nucleotides, 2'-fluoro nucleotides, deoxyribonucleotides, and ribonucleotides. In one embodiment, all nucleic acid linker nucleotides are the same type of nucleotide. In one embodiment, the nucleic acid linker entirely comprises 2'-O-methyl nucleotides, entirely comprises 2'-fluoro nucleotides, or entirely comprises deoxyribonucleotides.
  • the cleavable linking group (tether) comprises a polynucleotide comprising a modified ribonucleotide sequence, optionally a polynucleotide comprising one or more modifications selected from the group consisting of a 2'-O-methyl ribonucleotide modification, a 2'-fluoro-ribonucleotide modification, a 2'-5'-linked nucleotide with different 3'-modification (3' -ribo, 3'-O-methyl, 3' -deoxy, 3' -fluoro), a glycol nucleic acid (GNA) modification, a locked nucleic acid (LNA) modification, a hexanol nucleic acid (HNA) modification, an abasic ribose modification, an abasic deoxyribose modification, and an abasic hydroxyprolinol modification.
  • GAA glycol nucleic acid
  • LNA locked nucleic acid
  • HNA
  • the linking group L in the single-stranded oligonucleotide of formula (I) comprises a nucleotide-based cleavable linking group (tether) that is cleavable by DICER.
  • the single-stranded oligonucleotide comprises a substrate cleavable by DICER.
  • the single-stranded oligonucleotide contains a cleavable linking group (nucleotide-based or non-nucleotide-based) capable of generating a metabolite of a 5' -monophosphate at at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ) of the single- stranded oligonucleotide.
  • a cleavable linking group nucleotide-based or non-nucleotide-based
  • the single-stranded oligonucleotide may further comprise one or more ligands (e.g., targeting ligands).
  • Z 1 comprises at least one ligand (e.g., a targeting ligand), at the 5' or 3' end of the sequence.
  • Z 2 comprises at least one ligand (e.g., a targeting ligand), at the 5' or 3' end of the sequence.
  • each of Z 1 andZ 2 comprises at least one ligand (e.g., a targeting ligand), at the 5' or 3' end of the sequence.
  • At least one of the ligands is a lipophilic moiety.
  • the lipophilic moiety is lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, docosanoic acid (DCA), dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, lithocholic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
  • DCA docosanoic acid
  • the lipid is a fatty acid (an omega-3 fatty acid, for example), selected from the group consisting of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the lipophilic moiety contains a saturated or unsaturated C 4 -C 30 hydrocarbon chain (e.g., C 4 -C 30 alkyl or alkenyl), and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.
  • the lipophilic moiety contains a saturated or unsaturated C 6 -C 18 hydrocarbon chain (e.g., a linear C 6 -C 18 alkyl or alkenyl), e.g., a saturated or unsaturated C 16 hydrocarbon chain (e.g., a linear C 16 alkyl or alkenyl).
  • the lipophilic moiety contains a saturated or unsaturated C 14 -C 24 hydrocarbon chain (e.g., a linear C 14 -C 24 alkyl or alkenyl), e.g., a saturated or unsaturated C 22 hydrocarbon chain (e.g., a linear C 22 alkyl or alkenyl).
  • one or more non-terminal positions of the single-stranded oligonucleotide may have the following structure: (1), wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives), and the n-hexadecyl chain is the lipophilic moiety.
  • B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives)
  • the n-hexadecyl chain is the lipophilic moiety.
  • the modification shown in formula (1) is referred to herein as “2'-C 16 ''.
  • one or more non-terminal positions of the single- stranded oligonucleotide may have the following structure: (2), wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives), and the n-docosanyl chain is the lipophilic moiety.
  • B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives)
  • the n-docosanyl chain is the lipophilic moiety.
  • the modification shown in formula (2) is referred to herein as “2' -C 22 '''
  • one or more non-terminal nucleotide positions of at least one of Z 1 and Z 2 have the 2'-C 4 -C 30 hydrocarbon chain structure, 2'-C 6 -C 18 hydrocarbon chain structure, 2'-C 14 -C 24 hydrocarbon chain structure, 2'-C 16 structure of formula (1), or 2'- C 22 structure of formula (2).
  • one or more non-terminal nucleotide positions of both Z 1 and Z 2 have the 2'-C 4 -C 30 hydrocarbon chain structure, 2'-C 6 -C 18 hydrocarbon chain structure, 2'- C 14 -C 24 hydrocarbon chain structure, 2'- C 16 structure of formula (1), or 2'- C 22 structure of formula (2).
  • the lipophilic moiety contains one or more phospholipids.
  • the lipophilic moiety contains one or more lipids or lipophilic ligands disclosed in International PCT Application Publication Nos. WO 2019/232255A1 and WO 2021/108662A1, and U.S. Patent No. 10,184,124; all of which are herein incorporated by reference in their entirety.
  • the ligands include one or more of the following formulas:
  • n 1 in “C10-TEG- and n is 7 to “16-TEG- " . (L-4)
  • the ligands include those disclosed in International PCT
  • At least one of Z 1 and Z 2 comprises one or more lipophilic moieties conjugated independently to one or more of the internal positions (i.e., non-terminal positions) excluding positions 9-12 on a nucleotide sequence; for instance, positions 4-8 and 13-18 on a nucleotide sequence; positions 5, 6, 7, 15, and 17 on a nucleotide sequence; or positions 4, 6, 7, and 8 on a nucleotide sequence, each counting from the 5' -end of the nucleotide sequence as position 1.
  • At least one of Z 1 and Z 2 comprises one or more lipophilic moieties conjugated independently to position 6 of the nucleotide sequence, counting from the 5'-end of the nucleotide sequence.
  • each of Z 1 and Z 2 comprises a lipophilic moiety conjugated to position 6 of the nucleotide sequence; optionally the lipophilic moiety comprises a saturated or unsaturated C 6 -C 18 hydrocarbon chain, or a saturated or unsaturated C 14 -C 24 hydrocarbon chain; optionally the lipophilic moiety comprises a saturated or unsaturated C 16 hydrocarbon chain or a saturated or unsaturated C 22 hydrocarbon chain.
  • At least one of Z 1 and Z 2 comprises one or more lipophilic moieties conjugated independently to one or more of internal positions (i.e., non-terminal positions) on a nucleotide sequence; for instance, positions 6-10 and 15-18 on a nucleotide sequence; and positions 15 and 17 on a nucleotide sequence, each counting from the 5' -end of the nucleotide sequence as position 1.
  • At least one of the ligands is a targeting ligand selected from the group consisting of an antibody, antigen, folate, receptor ligand, carbohydrate, aptamer, integrin receptor ligand, chemokine receptor ligand, transferrin, biotin, serotonin receptor ligand, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligand.
  • at least one of the ligands is an integrin receptor ligand.
  • the targeting ligand may be conjugated to an internal position of a nucleotide sequence (e.g., Z 11 and Z 12 ), optionally via a linker or carrier.
  • the targeting ligand may be conjugated to the 3'-end or 5'-end of Z 11 or Z 12 , optionally via a linker or carrier.
  • the ligands is a carbohydrate-based ligand.
  • the carbohydrate-based ligand may be D-galactose, multivalent galactose, N-acetyl-D- galactosamine (GalNAc), multivalent GalNAc, D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent fucose, glycosylated polyaminoacids, or lectins.
  • the carbohydrate-based ligand is an ASGPR ligand.
  • the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trival ent branched linker, such as:
  • At least one of the ligands may be conjugated at the 3'- end, 5'-end, or an internal position of a nucleotide sequence (e.g., Z 1 and Z 2 ).
  • At least one of the ligands may be conjugated to the single- stranded oligonucleotide via a direct attachment to the ribosugar of the oligonucleotide.
  • the ligand may be conjugated to the single-stranded oligonucleotide via one or more linkers (tethers), and/or a carrier.
  • the ligand may be conjugated to the single-stranded oligonucleotide via a monovalent or branched bivalent or trivalent linker.
  • the ligand may be conjugated to the single-stranded oligonucleotide via a carrier that replaces one or more nucleotide(s).
  • the carrier can be a cyclic group or an acyclic group.
  • the cyclic group is selected from the group consisting of cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] di oxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.
  • the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.
  • the single-stranded oligonucleotide may be characterized by one or more of:
  • Z 1 and Z 2 each independently contain 19-23 optionally modified nucleotides
  • Q 1 and Q 2 each independently contain 0 to 2 optionally modified nucleotides
  • the duplexed region formed by Z 1 and Z 2 contains no more than 3 mismatched base pairs
  • At least one nucleotide in Z 2 is a modified nucleotide
  • At least one nucleotide in Z 1 is a modified nucleotide
  • Z 2 comprises at least one modified intemucleotide linkage
  • Z 1 comprises at least one modified intemucleotide linkage
  • the 5'-terminal nucleotide comprises a 5'-phosphate or 5'-phosphate mimic modification
  • the 3'-terminal nucleotide is conjugated to a ligand, optionally through a linker;
  • (k) Z 1 contains no more than 3 mismatches to the target gene
  • (l) Z 1 and Z 2 each independently contain 19-23 optionally modified nucleotides
  • (m) L contains a linking moiety represented by a formula: #-(N) n -**, wherein n is 3 to 5; and each N is independently an optionally modified nucleotide, Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, QI 1, Q150, Q151, Q173, Q221, Q222, Q367, or Q368.
  • the single-stranded oligonucleotide may be characterized by two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or all of the above features.
  • the single-stranded oligonucleotide may be characterized by one or more of
  • Z 1 and Z 2 each independently contain 21 optionally modified nucleotides
  • Q 1 and Q 2 each independently contain 2 optionally modified nucleotides
  • the duplexed region formed by Z 1 and Z 2 contains no more than 3 mismatched base pairs
  • (g) Z 2 comprises at least two consecutive modified internucleotide linkages
  • Z 1 comprises at least two consecutive modified internucleotide linkages
  • the 5'-terminal nucleotide of Z 1 comprises a 5'-phosphate or 5'-phosphate mimic modification
  • (k) Z 1 contains no more than 3 mismatches to the target gene
  • (l) L contains a linking moiety represented by a formula: #-(N) n -**, wherein n is 5; and each N is independently an optionally modified nucleotide, or Q304.
  • the single-stranded oligonucleotide may be characterized by two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or all of the above features.
  • Another aspect of the invention relates to a single-stranded oligonucleotide according to formula (II) or (III):
  • Z 11 is a first oligonucleotide, comprising 15 - 100 optionally modified nucleotides that is substantially complementary to a target gene;
  • Z 12 is a second oligonucleotide, comprising 10 - 100 optionally modified nucleotides that is substantially complementary to Z 11 ;
  • Z 11 and Z 12 are capable of forming an intra-strand duplexed region comprising 7 or more consecutive base pairs;
  • Q s represents 0 to 12 optionally modified nucleotides
  • L is an optional linking group; at least one nucleotide in formula (II) is a modified nucleotide; and at least one nucleotide in formula (III) is a modified nucleotide, wherein at least one nucleotide at the 3' end of Z 11 , for formula (II), at least one nucleotide at the 5' end of Z 11 , for formula (III), in either case together with L and Q s form a loop region connecting Z 11 and Z 12 .
  • the single-stranded oligonucleotide may be an inhibitory single- stranded oligonucleotide, such as an antisense oligonucleotide (ASO), an antimiR (antagomir) oligonucleotide, microRNA mimic, supermir, aptamer, U1 adaptor, triplex- forming oligonucleotide, RNA activator, immuno-stimulatory oligonucleotide, decoy oligonucleotide, heteroduplex-forming oligonucleotide, or a single-stranded siRNA (ss- siRNA) oligonucleotide.
  • ASO antisense oligonucleotide
  • antagomir antimiR
  • microRNA mimic microRNA mimic
  • supermir supermir
  • aptamer oligonucleotide
  • U1 adaptor aptamer
  • U1 adaptor aptamer
  • aptamer
  • the first oligonucleotide Z 11 and second oligonucleotide Z 12 each may independently comprise 10 - 100 optionally modified nucleotides.
  • Z 11 and Z 12 each may independently comprise 10 - 40, 10 - 30, 12 - 26, 12 - 23, 12 - 21, 15 - 26, 15 - 23, 15-21, 19 - 26, 19 - 23, or 19 - 21 optionally modified nucleotides.
  • the first oligonucleotide Z 11 and second oligonucleotide Z 12 each may independently comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • Z 11 and Z 12 each may independently have about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 to about 26 nucleotides, about 10 to about 23 nucleotides, about 10 to about 21 nucleotides, about 12 to about 50 nucleotides, about 12 to about 40 nucleotides, about 12 to about 35 nucleotides, about 12 to about 30 nucleotides, about 12 to about 26 nucleotides, about 12 to about 23 nucleotides, about 12 to about 21 nucleotides, about 15 to about 50 nucleotides, about 15 to about 40 nucleotides, about 15 to about 35 nucleotides, about 15 to about 30 nucleotides, about 15 to about 26 nucleotides, about 15 to about 23 nucleotides, about 15 to about 21 nucleotides, about 19 to about 50 nucleotides, about 19 to
  • Z 11 and Z 12 each independently comprise 10 - 40 optionally modified nucleotides. In some embodiments, Z 11 and Z 12 each independently comprise 12 - 26 optionally modified nucleotides.
  • Z 11 and Z 12 each contain the same number of optionally modified nucleotides. In some embodiments, Z 11 contain a larger number of optionally modified nucleotides than Z 12 . In some embodiments, Z 11 comprises 19 - 26 optionally modified nucleotides, and Z 12 comprises 12-21 optionally modified nucleotides.
  • the single-stranded oligonucleotide can be cleaved at the linking group L.
  • the first oligonucleotide Z 11 can be cleaved into an antisense strand that is substantially complementary to a target gene (e.g., a target mRNA or DNA), and the second oligonucleotide Z 12 can be cleaved into a sense strand that is substantially complementary to Z 11 .
  • Q s may comprise 0 to 12 optionally modified nucleotides.
  • Q s may comprise 0 to 10, 0 to 6, 0 to 4, 0 to 3, 0 to 2, 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3 optionally modified nucleotides.
  • Q s is 0.
  • Q s is 1 to 6 optionally modified nucleotides.
  • Q s is 2 optionally modified nucleotides.
  • Q s is 1 optionally modified nucleotide.
  • one or more nucleotides of Q s form a mismatched base pair with the opposite nucleotide in Z 11 .
  • Q s is 2 optionally modified nucleotides, and is characterized by one of the followings: both nucleotides of Q s form mismatched base pairs with their opposite nucleotides in Z 11 , one nucleotide of Q s forms a mismatched base pair with the opposite nucleotide in Z 11 (e.g., the nucleotide of Q s next to Z 12 forms a mismatched base pair with the opposite nucleotide in Z 11 ), or both nucleotides of Q s form base pairs with their opposite nucleotides in Z 11 .
  • Q s is two 2'-deoxy modified nucleotides.
  • Q s is -dTdT-.
  • the first oligonucleotide Z 11 is substantially complementary to a target gene, i.e., Z 11 contains no more than 3 (e.g., 0, 1, 2, or 3) mismatches to the target gene.
  • the target gene may be a mRNA, pre-mRNA, microRNA, pre-miRNA, long non-coding RNA (IncRNA), or DNA.
  • the first oligonucleotide Z 11 and the second oligonucleotide Z 12 are capable of forming an intra-strand duplexed region, e.g., comprising 7 or more consecutive base pairs.
  • Z 11 and Z 12 are capable of forming an intra-strand duplexed region comprising 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 base pairs.
  • Z 11 and Z 12 are capable of forming an intra-strand duplexed region having base pairs with all the nucleotides of Z 12 .
  • the intra-strand duplexed region formed by Z 11 and Z 12 may contain all consecutive base pairs, or may contain up to 3 mismatch based pairs (e.g., 0, 1, 2, or 3). In some embodiments, the intra-strand duplexed region formed by Z 11 and Z 12 contain 1 mismatch based pair.
  • the first oligonucleotide Z 11 and the second oligonucleotide Z 12 are capable of forming an intra-strand duplexed region at the seed region of Z 11 (e.g., the seed region of an antisense strand; e.g., at positions 2-8 of the 5'-end of the antisense strand).
  • the first oligonucleotide Z 11 contains a loop at the 3' -end or 5'-end.
  • the first oligonucleotide Z 11 comprises W — LP, wherein W is capable of forming an intra-strand duplexed region of at least 7 base pairs with Z 12 , and LP, optionally together with L, forms the loop between W and Z 12 at the 3' -end or 5' -end.
  • W and Z 12 are capable of forming an intra-strand duplexed region comprising 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 base pairs.
  • W and Z 12 are capable of forming an intra-strand duplexed region having base pairs with all the nucleotides of Z 12 .
  • the intra-strand duplexed region formed by W and Z 12 may contain all consecutive base pairs, or may contain no more than 3 (e.g., 0, 1, 2, or 3) mismatch based pairs.
  • the single-stranded oligonucleotide is represented by formula (Ila) or formula (Illa): wherein:
  • Z 11 comprises W — LP
  • W forms an intra-strand duplexed region at least 7 base pairs with Z 12 ,
  • LP optionally together with L, forms a loop between W and Z 12 at the 3' -end or 5'- end, re p rese nt s an optional presence of L, represents an optional presence of Q s , represents an optional overhang at 5'-end or 3'-end of Z 11 , and represents an optional overhang at 5' -end or 3' -end of Z 12 .
  • the duplexed region formed by Z 11 and Z 12 at the non-loop terminal has a blunt end. In some embodiments, the duplexed region formed by W and Z 12 at the non-loop terminal has a blunt end.
  • Z 11 at the non-loop terminal has an overhang of 1-3 nucleotides in length.
  • W at the non-loop terminal has an overhang of
  • 1-3 nucleotides in length In some embodiments, is present and is 1-3 nucleotides in length.
  • Z 12 has an overhang of 1-3 nucleotides in length. In some embodiments, is present and is 1-3 nucleotides in length.
  • the overhang is 1 nucleotide in length. In some embodiments, the overhang is 2 nucleotides in length. In some embodiments, the overhang is 3 nucleotides in length.
  • Each of the nucleotides in the single-stranded oligonucleotide may be independently and optionally modified.
  • Each of the nucleotides in first oligonucleotide Z 11 and second oligonucleotide Z 12 may be independently and optionally modified.
  • the single-stranded oligonucleotide comprises at least one chemical modification.
  • each of the first oligonucleotide Z 11 and second oligonucleotide Z 12 comprise at least one chemical modification.
  • W comprises at least one chemical modification.
  • all the nucleotides in Z 11 are modified nucleotides.
  • all the nucleotides in W are modified nucleotides.
  • all the nucleotides in Z 12 are modified nucleotides.
  • all the nucleotides of the single-stranded oligonucleotide are modified.
  • the chemical modification to the nucleotide(s) may include an intemucleoside linkage modification, a nucleobase modification, a sugar modification, or combinations thereof.
  • the chemical modification is selected from the group consisting of LNA, ENA, HNA, CeNA, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'- O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-O-alkyl, 2'-O-allyl, 2'-C- allyl, 2'-fluoro, 2' -deoxy, 2'-O-N-methylacetamido ( 2'-O-NMA), 2'-O-dimethylaminoethoxy ethyl (2'-O- DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modification (such as 2'- modified L-nucleoside, e.g., 2' -deoxy -L-nucleoside), BNA abasic sugar,
  • the chemical modification is selected from the group consisting of at least one of the modified nucleotides is a deoxy-nucleotide, a 3 '-terminal deoxythimidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide (LNA), an unlocked nucleotide (UNA), a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, 2'-C- alkyl-modified nucleotide, 2' -hydroxy-modified nucleotide, a 2' -methoxy ethyl
  • dT deoxy-
  • the chemical modification is a 2' -modification selected from the group consisting of 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O- methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, 2'-fluoro, and combinations thereof.
  • about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 11 are modified.
  • about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of Z 12 are modified.
  • about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of all the nucleotides in the single-stranded oligonucleotide are modified. For example, when 50% of all the nucleotides are modified, 50% of all nucleotides present in the single-stranded oligonucleotide contain at least one modification as described herein.
  • At least 50% of the nucleotides of the single-stranded oligonucleotide are independently modified with 2'-O-methyl, 2'-O-allyl, 2'-O-methoxyalkyl (e.g., 2'-O-methoxymethyl, 2'-O-methoxyethyl, or 2'-O-2-methoxypropanyl), 2'-deoxy, or 2' -fluoro.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) further comprises one or more of the following internucleotide linkage modifications;
  • one or more internucleotide linkages among the six 3 '-terminal nucleotides is a modified intemucleotide linkage
  • one or more internucleotide linkages among the six 5 '-terminal nucleotides is a modified intemucleotide linkage.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) further comprises one or more intemucleotide linkages among the eight 3'-terminal nucleotides of Z 11 for formula (II), or one or more intemucleotide linkages among the eight 5'-terminal nucleotides of Z 11 for formula (III), is a modified intemucleotide linkage.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) further comprises one or more of the following intemucleotide linkage modifications:
  • the single-stranded oligonucleotide when the single-stranded oligonucleotide contains a terminal conjugation of a ligand to the 5'-end or 3 '-end nucleotide, or contains a terminal conjugation of an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide to the 5'-end or 3 '-end nucleotide, then at that terminus, the above intemucleotide linkage modifications to the terminal nucleotide can be omitted.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) further comprises one of the following intemucleotide linkage modifications:
  • one or more intemucleotide linkages among the eight 3 '-terminal nucleotides of Z 11 for formula (II) (or Ila), or one or more intemucleotide linkages among the eight 5'- terminal nucleotides of Z 11 for formula (III) (or (Illa)), is a modified intemucleotide linkage;
  • one or more intemucleotide linkages among the six 5'-terminal nucleotides of Z 11 for formula (II) (or Ila), or one or more intemucleotide linkages among the six 3 '-terminal nucleotides of Z 11 for formula (III) (or (Illa)), is a modified intemucleotide linkage;
  • one or more intemucleotide linkages among the six 3 '-terminal nucleotides of Z 12 for formula (II) (or (Ila)), or one or more intemucleotide linkages among the six 5'-terminal nucleotides of Z 12 for formula (III) (or (Illa)), is a modified intemucleotide linkage.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa)) further comprises one or more of the following intemucleotide linkage modifications:
  • the single-stranded oligonucleotide of formula (II) (or Ila) further comprises one or more modified internucleotide linkage between the 5'-end nucleotide of Z 12 and the first nucleotide of Q s .
  • the single-stranded oligonucleotide of formula (III) (or (Illa)) further comprises one or more modified intemucleotide linkage between the 3'-end nucleotide of Z 12 and the first nucleotide of Q s .
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa)) further comprises one or more modified intemucleotide linkages between the nucleotides of Q s .
  • the modified intemucleotide linkage is phosphorothioate linkage.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) comprises at least two consecutive phosphorothioate or methylphosphonate intemucleotide linkage modifications. In some embodiments, the single- stranded oligonucleotide comprises at least two blocks of two consecutive phosphorothioate or methylphosphonate intemucleotide linkage modifications. In some embodiments, the single- stranded oligonucleotide comprises at least three blocks of two consecutive phosphorothioate or methylphosphonate intemucleotide linkage modifications.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) has at least two phosphorothioate intemucleotide linkages among the first six nucleotides on a nucleotide sequence (e.g., Z 11 and/or Z 12 ).
  • a nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 11 and/or Z 12 ) comprises two blocks of one, two, or three phosphorothioate intemucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate intemucleotide linkages.
  • a nucleotide sequence of the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) (e.g., Z 11 and/or Z 12 ) comprises at least two consecutive phosphorothioate intemucleotide linkage modifications within positions 18-23 of the nucleotide sequence, counting from the 5 '-end of the nucleotide sequence.
  • a nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 11 and/or Z 12 ) comprises at least two consecutive phosphorothioate intemucleotide linkage modifications within position 1-5 of the nucleotide sequence, counting from the 5 '-end of the nucleotide sequence.
  • each of Z 11 and Z 12 of the single-stranded oligonucleotide comprises at least two consecutive phosphorothioate intemucleotide linkage modifications.
  • each of Z 11 and Z 12 of the single-stranded oligonucleotide comprises: at least two consecutive phosphorothioate internucleotide linkage modifications within positions 18-23 of the nucleotide sequence, and at least two consecutive phosphorothioate intemucleotide linkage modifications within position 1-5 of the nucleotide sequence, counting from the 5 '-end of the nucleotide sequence.
  • Z 11 at the non-loop terminal has an overhang of 1-3 nucleotides in length. In one embodiment, Z 11 at the non-loop terminal has an overhang of 2 nucleotides in length (e.g., at the 3'-end of Z 11 ) and has a phosphorothioate intemucleotide linkage between the two overhang nucleotides.
  • Z 11 at the non-loop terminal has an overhang of 2 nucleotides in length and has two phosphorothioate intemucleotide linkages between the terminal 3 nucleotides (e.g., at the 3'-end of Z 11 ), in which 2 of the 3 nucleotides are the overhang nucleotides, and the third is the paired nucleotide next to the overhang nucleotide.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) further comprises a phosphate or phosphate mimic at the 5'-end of a nucleotide sequence (e.g., Z 11 and/or Z 12 ).
  • the single-stranded oligonucleotide comprises a phosphate mimic at the 5'-end of a nucleotide sequence (e.g., Z 11 and/or Z 12 ).
  • at least one phosphate mimic is at the 5' end of Z 11 .
  • the phosphate mimic is a 5 '-vinyl phosphonate (VP).
  • the phosphate mimic is a 5 '-cyclopropyl phosphonate.
  • the phosphate mimic is a 5 '-vinyl phosphate.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) further comprises at least one terminal, chiral phosphorus atom.
  • the 5 -'end or 3 -'end nucleotide in the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) comprise a 2'-5'-linked nucleotide modification; or the 5'-end or 3'-end nucleotide is conjugated to an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide (e.g., ribonucleotide), optionally via a phosphodiester, phosphorothioate, or phosphodithioate linkage.
  • the 5 -'end or 3 -'end nucleotide in the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa) is modified to comprise a linking moiety containing a mono-, di-, tri-, tetra-, penta- or polyprolinol, or mono-, di-, tri-, tetra-, penta- or polyhydroxyprolinol.
  • At least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5' -end or the 3' -end of the single-stranded oligonucleotide are not phosphorothioate linkages.
  • the at least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5'-end or the 3'-end of the single-stranded oligonucleotide are each independently a natural phosphate group or a phosphodiester linkage, or a PN-linkage.
  • At least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5' -end or the 3' -end of the single-stranded oligonucleotide are PN-linkages comprising an optionally substituted cyclic guanidine moiety, for instance, those having the structure of , wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S.
  • At least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5' -end or the 3' -end of the single-stranded oligonucleotide are PN-linkages comprising a triazole moiety (e.g., an optionally substituted triazolyl group), such as those having the structure of wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S.
  • At least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5' -end or the 3' -end of the single-stranded oligonucleotide are PN-linkages comprising an alkyne moiety (e.g., an optionally substituted alkynyl group), such as those having the structure of , wherein W is O or S. In some embodiments, W is O.
  • W is S.
  • At least one, two, three, four, or each of the five terminal phosphorous-containing linkages of the 5' -end or the 3' -end of the single-stranded oligonucleotide are PN-linkages comprising a Tmg group ( ), J such as those having the structure of wherein W is O or S. In some embodiments, W is O. In some embodiments, W is S.
  • the PN-linkage may be stereochemically controlled.
  • the 3-5 terminal nucleotides of Z 11 in the single-stranded oligonucleotide formula (II) (or Ila) or formula (III) (or (Illa), contain modifications selected from the group consisting of 2' -deoxy nucleotide (dN), a 2'- deoxy -2' -fluoronucleotide (fN), a ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'- aranucleotide (aN).
  • dN 2' -deoxy nucleotide
  • fN 2'- deoxy -2' -fluoronucleotide
  • rN ribonucleotide
  • mN 2'-O-methylnucleotide
  • aN 2'- aranucleotide
  • the 3 terminal nucleotides of Z 11 connected to L, Q s , or Z 12 , have modifications selected from the group consisting of:
  • # is the bond to Z 11 and ** is the bond to L, Q s , or Z 12 , dN represents a 2' -deoxy nucleotide, fN represents a 2' -deoxy-2' -fluoro nucleotide, rN represents a ribonucleotide, and mN represents a 2'-O-methyl nucleotide.
  • the 5 terminal nucleotides of Z 11 connected to L, Q s , or Z 12 , have modifications selected from the group consisting of:
  • # is the bond to Z 11 and ** is the bond to L, Q s , or Z 12
  • dN represents a 2' -deoxy nucleotide
  • fN represents a 2' -deoxy-'2 -fluoro nucleotide
  • rN represents a ribonucleotide
  • mN represents a 2'-O-methyl nucleotide.
  • LP and/or L together with L and/or Q s , connected to Z 12 contains modifications #-dN-dN-rN-rN-rN-dN-dN-**, wherein:
  • # is the bond to Z 11 and ** is the bond to Z 12
  • dN represents a 2' -deoxy nucleotide
  • rN represents a ribonucleotide (e.g., uridine or 5-methyluridine).
  • LP and/or L together with L and/or Q s , connected to Z 12 contains modifications #-dT-dT-rN-rN-rN-dN-dN-**, wherein:
  • # is the bond to Z 11 and ** is the bond to Z 12
  • dN represents a 2' -deoxy nucleotide
  • rN represents a ribonucleotide (e.g., uridine or 5-methyluridine).
  • LP and/or L together with L and/or Q s , connected to Z 12 contains modifications #-dN-dN-rN-rN-rN-dT-dT-**, wherein:
  • # is the bond to Z 11 and ** is the bond to Z 12
  • dN represents a 2' -deoxy nucleotide
  • rN represents a ribonucleotide (e.g., uridine or 5-methyluridine).
  • LP and/or L together with L and/or Q s , connected to Z 12 contains modifications #-dT-dT-rN-rN-rN-dT-dT-**, wherein:
  • # is the bond to Z 11 and ** is the bond to Z 12
  • dN represents a 2' -deoxy nucleotide
  • rN represents a ribonucleotide (e.g., uridine or 5-methyluridine).
  • the first oligonucleotide Z 11 contains at least one motif of three consecutive 2'-O-methyl modifications at positions 11, 12, and 13 from the 5'-end of Z 11 , and the nucleotide next to the motif is not 2'-O-methyl modified.
  • the second oligonucleotide Z 12 optionally together with Q s , contains at least one motif of three consecutive 2'-F modifications, and the nucleotide next to the motif is not 2'-F modified.
  • the position of the motif of three consesutive modifications is characterized by one or the followings: the motif is at Q s , positions 1 and 2 of Z 12 , optionally Z 11 is 19 nucleotides in length; the motif is at positions 1, 2, and 3 of Z 12 , optionally Z 11 is 20 nucleotides in length; the motif is at positions 2, 3, and 4 of Z 12 , optionally Z 11 is 21 nucleotides in length; the motif is at positions 3, 4, and 5 of Z 12 , optionally Z 11 is 22 nucleotides in length; or the motif is at positions 4, 5, and 6 of Z 12 , optionally Z 11 is 23 nucleotides in length.
  • Z 12 optionally together with Q s , contains a 2'-O-methyl or 2'-F modification at a position that is 2 positions before the motif (position n-2, if the motif starts at position n), provided that the position is not part of Z 11 .
  • L is a cleavable linking group.
  • the cleavable linking group is cleavable in a homogenate, tritosome, cytosol, or endosome of any types of cells.
  • the cleavable linking group may be cleavable in liver homogenates, liver tritosomes, liver lysosomes, liver cytosol, liver endosome, brain homogenates, brain tritosomes, brain lysosomes, brain cytosol, or brain endosome.
  • the cleavable linking group is a redox cleavable linker (such as a reductively cleavable linker; e.g., a disulfide group), an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavable linker (e.g., an ester group), a phosphatase cleavable linker (e.g., an ester group), a peptidase cleavable linker (e.g., an ester group), or endosomal cleavable linker (or a protease cleavable linker, e.g., a carbohydrate linker).
  • a redox cleavable linker such as a reductively cleavable linker; e.g., a disulfide group
  • L is present in formula (II) (or Ila) or formula III (or Illa), and contains a linking moiety represented by a formula: #-(N) n -**.
  • # is the bond to Z 11 and ** is the bond to Q s or Z 12 ;
  • n is 3 to 12; and each N is independently a linking monomer having a chain length of 3 or more atoms.
  • each N may be independently a linking monomer having a chain length of 3 or more atoms.
  • the “chain length'' has been defined herein above.
  • n is 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5.
  • n is 3.
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) may be an optionally modified nucleotide.
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) may be independently selected from the group consisting of a 2'- deoxynucleotide (dN), a 2 '-deoxy-2' -fluoro nucleotide (fN), a ribonucleotide (rN), 2'-O- methylnucleotide (mN), and 2'-ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara- 2'-OMe, or 2'-ara ribonucleotide).
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) may contain a modified internucleotide linkage selected from the group consisting of a phosphodiester, phosphotriester (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), methylenemethylimino, a nitrogen-modified phosphorous-containing linkage (PN-linkage) (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), thiodiester, thionocarbamate, N,N'-dimethylhydrazine, phosphoroselenate, borano phosphat
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) may contain a moiety selected from the group consisting of an aliphatic saturated or unsaturated alkyl chain; a phosphorous-containing linkage, including a phosphate, a phosphonate, a phosphoramidate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphodiester, phosphotriester (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), and a nitrogen-modified phosphorous- containing linkage (PN-linkage) (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration); a (poly)ethylene glycol chain, including diethylene glycol, triethylene glycol, tetra, penta
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) may contain a moiety selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) may be independently selected from the group consisting of: wherein:
  • Base is an optionally modified nucleobase
  • R D is a C 4-30 alkyl, C 4-30 alkyenyl, or C 4-30 alkynyl.
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) comprise a mono-, di-, tri-, tetra-, penta- or poly-prolinol, optionally conjugated with a ligand; a mono-, di-, tri-, tetra-, penta- or poly-hydroxyprolinol, optionally conjugated with a ligand; an optionally modified nucleotide; or combinations thereof.
  • L of formula (II) (or Ila) or formula III (or Illa) contains one or more of a mono-, di-, tri-, tetra-, penta- or poly-prolinol, optionally conjugated with a ligand; and one or more optionally modified nucleotides.
  • L of formula (II) (or Ila) or formula III (or Illa) contains one or more of a mono-, di-, tri-, tetra-, penta- or poly-hydroxyprolinol, optionally conjugated with a ligand; and one or more optionally modified nucleotides.
  • one or more linking moieties (N) in L of formula (II) (or Ila) or formula III (or Illa) may be independently selected from the group consisting of Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, QI 1, Q150, Q151, Q173, Q221, Q222, Q367, and Q368.
  • each linking moiety (N) in L of formula (II) (or Ila) or formula III (or Illa) is independently an optionally modified nucleotide, Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, QI 1, Q150, Q151, Q173, Q221, Q222, Q367, or Q368.
  • L of formula (II) (or Ila) or formula III (or Illa) contains 3- 5 of 2'-deoxy nucleotides, a triplet of 2 '-deoxy-2' -fluoro nucleotides, a triplet of ribonucleotides, a triplet of 2'-O-methyl nucleotides, or a triplet of Q304. In one embodiment, L contains a triplet of Q304.
  • the position of L in formula (II) (or Ila) or formula III (or Illa) is characterized by one of the followings: all the linking monomer of L, together with LP, form a loop between W and Z 12 ; one or more of the linking monomers of L, together with LP, forms a loop between W and Z 12 , and one or more of the linking monomers of L is not in the loop region; one or more of the linking monomers of L, together with LP, forms a loop between W and Z 12 , and one or more of the linking monomers of L is not in the loop and is connected to Q s ; and one or more of the linking monomers of L, together with LP, forms a loop between W and Z 12 , and one or more of the linking monomers of L is not in the loop and is connected to Z 12 .
  • one or more internucleotide linkages between the nucleotides in L of formula (II) (or Ila) or formula III (or Illa) may be modified intemucleotide linkages independently selected from the group consisting of a phosphodiester, phosphotriester (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), phosphorothioate (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration), and a nitrogen-modified phosphorous-containing linkage (PN-linkage) (optionally comprising the linkage phosphorus atom in either Rp configuration or Sp configuration).
  • PN-linkage nitrogen-modified phosphorous-containing linkage
  • L of formula (II) (or Ila) or formula III (or Illa) may contain one or more linking moiety selected from the group consisting of a triazole linkage, an amide linkage, a sulfide or disulfide linkage, a phosphate linkage, an oxime linkage, a hydrazo linkage, a N,N'-dialkylenehydrazo linkage, a methyleneimino linkage, a methylenecarbonylamino linkage, a methylenemethylimino linkage, a methylenehydrazo linkage, a methylenedimethylhydrazo linkage, a methyleneoxymethylimino linkage, a hydroxylamino linkage, a formacetal linkage, an alkyl or aryl linkage, a PEG linkage, an ether linkage, a thioether linkage, a thiodiester linkage, a thionocarbamate linkage,
  • L of formula (II) (or Ila) or formula III (or Illa) may contain one or more cyclic groups selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,
  • L of formula (II) (or Ila) or formula III (or Illa) contains a nucleotide-based linker (tether). In some embodiments, L contains a non-nucleotide-based linker (tether).
  • the nucleotide-based or non-nucleotide-based linker (tether) contained in L of formula (II) (or Ila) or formula III (or Illa) is a stable linker (tether) that is stable in a biological fluid.
  • the nucleotide-based or non-nucleotide based stable linker (tether) is stable in plasma or artificial cerebrospinal fluid.
  • the cleavable linking group (tether) comprises a moiety of formula (CL-1) or (CL-2), as described above.
  • the cleavable linking group comprises a moiety selected from the following:
  • n 0 or 1-20;
  • n 0 or 1-20; mono-, di-, tri-, tetra-, penta- or polyprolinol, optionally conjugated with a ligand; mono-, di-, tri-, tetra-, penta- or polyhydroxyprolinol, optionally conjugated with a ligand.
  • the cleavable linking group (tether) comprises a nucleic acid linker of 1 to 15 nucleotides in length.
  • the nucleic acid linker may be 2 to 7, 5 to 7, 2 to 5, or 3, 4, or 5 optionally modified nucleotides in length.
  • the cleavable linking group comprises a nucleic acid linker comprising one or more nucleotides selected from the group consisting of 2'-O- methyl nucleotides, 2'-fluoro nucleotides, deoxyribonucleotides, and ribonucleotides. In one embodiment, all nucleic acid linker nucleotides are the same type of nucleotide. In one embodiment, the nucleic acid linker entirely comprises 2'-O-methyl nucleotides, entirely comprises 2'-fluoro nucleotides, or entirely comprises deoxyribonucleotides.
  • the cleavable linking group (tether) comprises a polynucleotide comprising a modified ribonucleotide sequence, optionally a polynucleotide comprising one or more modifications selected from the group consisting of a 2'-O-methyl ribonucleotide modification, a 2'-fluoro-ribonucleotide modification, a 2'-5'-linked nucleotide with different 3' -modification (3' -ribo, 3'-O-methyl, 3' -deoxy, 3' -fluoro), a glycol nucleic acid (GNA) modification, a locked nucleic acid (LNA) modification, a hexanol nucleic acid (HNA) modification, an abasic ribose modification, an abasic deoxyribose modification, and an abasic hydroxyprolinol modification.
  • GAA glycol nucleic acid
  • LNA locked nucleic acid
  • the linking group L in the single-stranded oligonucleotide of formula (II) (or Ila) or formula III (or Illa) comprises a nucleotide-based cleavable linking group (tether) that is cleavable by DICER.
  • the single-stranded oligonucleotide comprises a substrate cleavable by DICER.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula III (or Illa) contains a cleavable linking group (nucleotide-based or non- nucleotide-based) capable of generating a metabolite of a 5'-monophosphate at at least one nucleotide sequence (e.g., Z 11 and/or Z 12 ) of the single-stranded oligonucleotide.
  • the single-stranded oligonucleotide of formula (II) (or Ila) or formula III (or Illa) may further comprise one or more ligands (e.g., targeting ligands).
  • Z 11 comprises at least one ligand (e.g., a targeting ligand), at the 5' or 3' end of the sequence.
  • Z 12 comprises at least one ligand (e.g., a targeting ligand), at the 5' or 3' end of the sequence.
  • each of Z 11 andZ 12 comprises at least one ligand (e.g., a targeting ligand), at the 5' or 3' end of the sequence.
  • At least one of the ligand is conjugated to an internal position of a nucleotide sequence (e.g., Z 11 and Z 12 ), optionally via a linker or carrier. In some embodiments, at least one of the ligand is conjugated to the 3'-end or 5'-end of Z 11 or Z 12 , optionally via a linker or carrier. In some embodiments, at least one of the ligands may be conjugated to the single-stranded oligonucleotide via a direct attachment to the ribosugar of the oligonucleotide. Alternatively, the ligand may be conjugated to the single-stranded oligonucleotide via one or more linkers (tethers), and/or a carrier.
  • tethers linkers
  • the internal position may refer to one of the positions 1-4 nucleotides upstream or downstream of the Q s . In some embodiments, the internal position may refer to one of the positions 1-4 nucleotides upstream or downstream of nucleotides of Z 12 paired to positions 11, 12, and 13 from the 5 '-end of Z 11 .
  • the terminal nucleotide (for Z 12 ) that is connected to Q s may be considered as internal positions.
  • the internal position may be characterized by: excluding the nucleotide of Q s and/or Z 12 that is directly connected to the loop region; and/or excluding position 2 or 14 from the 5'-end of Z 11 ; and/or excludes positions 11, 12, and 13 from the 5'-end of Z 11 ; and/or excluding the positions of Q s and/or Z 12 paired to positions 11, 12, and 13 from the 5'- end of Z 11 ; and/or excluding the two or three terminal positions from the 3'-end of Z 12 and the 5'-end of Z 11 for formula (II) or (Ila); and/or excluding the two or three terminal positions from the 5 '-end of Z 12 and the 3 -e'nd of Z 11 for formula (III) or (Illa).
  • the ligand may be conjugated to the single-stranded oligonucleotide via a monovalent or branched bivalent or trivalent linker.
  • the ligand may be conjugated to the single-stranded oligonucleotide via a carrier that replaces one or more nucleotide(s).
  • the carrier can be a cyclic group or an acyclic group.
  • the cyclic group is selected from the group consisting of cyclohexyl, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] di oxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.
  • the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.
  • at least one of the ligands comprises a lipophilic moiety.
  • the lipophilic moiety is lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, docosanoic acid (DC A), dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, lithocholic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dime
  • the lipid is a fatty acid (an omega-3 fatty acid, for example), selected from the group consisting of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the lipophilic moiety contains a saturated or unsaturated C 4 -C 30 hydrocarbon chain (e.g., C 4 -C 30 alkyl or alkenyl), and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.
  • the lipophilic moiety contains a saturated or unsaturated C 6 -C 18 hydrocarbon chain (e.g., a linear C 6 -C 18 alkyl or alkenyl), e.g., a saturated or unsaturated C 16 hydrocarbon chain (e.g., a linear C 16 alkyl or alkenyl).
  • the lipophilic moiety contains a saturated or unsaturated C 14 -C 24 hydrocarbon chain (e.g., a linear C 14 -C 24 alkyl or alkenyl), e.g., a saturated or unsaturated C 22 hydrocarbon chain (e.g., a linear C 22 alkyl or alkenyl).
  • one or more non-terminal positions of the single-stranded oligonucleotide may have a “2'-C 16 '' modification of formula (1), as described herein above, wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives), and the n-hexadecyl chain is the lipophilic moiety.
  • B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives)
  • the n-hexadecyl chain is the lipophilic moiety.
  • one or more non-terminal positions of the single- stranded oligonucleotide may have “2'-C 22 '' modification of formula (2), as described herein above, wherein B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives), and the n-docosanyl chain is the lipophilic moiety.
  • B is a natural or modified nucleotide base (e.g., adenine, guanine, cytosine, thymine or uracil, or their modified derivatives)
  • the n-docosanyl chain is the lipophilic moiety.
  • one or more non-terminal nucleotide positions of at least one of Z 11 and Z 12 have the 2'-C 4 -C 30 hydrocarbon chain structure, 2'-C 6 -C 18 hydrocarbon chain structure, 2'-C 14 -C 24 hydrocarbon chain structure, 2'-C 16 structure of formula (1), or 2'- C 22 structure of formula (2).
  • the lipophilic moiety contains one or more phospholipids.
  • the lipophilic moiety contains one or more lipids or lipophilic ligands disclosed in International PCT Application Publication Nos. WO 2019/232255A1 and WO 2021/108662A1, and U.S. Patent No. 10,184,124; all of which are herein incorporated by reference in their entirety.
  • the ligands include one or more of ligands of formulas (L- 1), (L-2), (L-3), or (L-4), as described herein above.
  • the ligands include those disclosed in International PCT Application Publication Nos. WO2017/053999, WO2019/118916, WO2022/031433, WO2022/056269, WO2022/056273, and WO2022/056277; all of which are herein incorporated by reference in their entirety.
  • the lipophilic moiety comprises a saturated or unsaturated C 4 -C 30 (e.g., C 4 -C 18 ) hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboylic acid, sulfonate, phostate, thiol, azide, and alkyne.
  • C 4 -C 30 e.g., C 4 -C 18
  • an optional functional group selected from the group consisting of hydroxyl, amine, carboylic acid, sulfonate, phostate, thiol, azide, and alkyne.
  • the lipophilic moiety is conjugated to one or more of the internal positions on Z 11 or Z 12 , optionally via a linker or carrier.
  • At least one of Z 11 and Z 12 comprises one or more lipophilic moieties conjugated independently to one or more of the internal positions (i.e., non-terminal positions) excluding positions 9-12 on a nucleotide sequence; for instance, positions 4-8 and 13-18 on a nucleotide sequence; positions 5, 6, 7, 15, and 17 on a nucleotide sequence; or positions 4, 6, 7, and 8 on a nucleotide sequence, counting from the 5 '-end of the nucleotide sequence as position 1.
  • At least one of Z 11 and Z 12 comprises one or more lipophilic moieties conjugated independently to position 6 of the nucleotide sequence, counting from the 5'-end of the nucleotide sequence.
  • each of Z 11 and Z 12 comprises a lipophilic moiety conjugated to position 6 of the nucleotide sequence; optionally the lipophilic moiety comprises a saturated or unsaturated C 4 -C 30 (e.g., C 4 -C 18 ) hydrocarbon chain, or a saturated or unsaturated C 14 -C 24 hydrocarbon chain; optionally the lipophilic moiety comprises a saturated or unsaturated C 16 hydrocarbon chain or a saturated or unsaturated C 22 hydrocarbon chain.
  • At least one of Z 11 and Z 12 comprises one or more lipophilic moieties conjugated independently to one or more of non-terminal positions on a nucleotide sequence; for instance, positions 6-10 and 15-18 on a nucleotide sequence; and positions 15 and 17 on a nucleotide sequence, counting from the 5 '-end of the nucleotide sequence as position 1.
  • At least one lipophilic moiety is conjugated to an internal position the single-stranded oligonucleotide of formula (II) (or Ila) or formula (III) (or (Illa)), wherein the internal position: excludes position 2 or 14 from the 5'-end of Z 11 ; and/or excludes positions 11, 12, and 13 from the 5'-end of Z 11 ; and/or excludes the positions of Q s and/or Z 12 paired to positions 11, 12, and 13 from the 5 -'end of Z 11 ; and/or optionally excludes the two or three terminal positions from the 3 '-end of Z 12 and the 5'- end of Z 11 for formula (II) or (Ila); and/or optionally excludes the two or three terminal positions from the 5'-end of Z 12 and the 3'- end of Z 11 for formula (III) or (Illa).
  • At least one of the ligands is a targeting ligand selected from the group consisting of an antibody, antigen, folate, receptor ligand, carbohydrate, aptamer, integrin receptor ligand, chemokine receptor ligand, transferrin, biotin, serotonin receptor ligand, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligand.
  • at least one of the ligands is an integrin receptor ligand.
  • the targeting ligand may be conjugated to an internal position of a nucleotide sequence (e.g., Z 11 and Z 12 ), optionally via a linker or carrier.
  • the targeting ligand may be conjugated to the 3'-end or 5'-end of Z 11 or Z 12 , optionally via a linker or carrier.
  • At least one of the ligands is a carbohydrate-based ligand.
  • the carbohydrate-based ligand may be D-galactose, multivalent galactose, N-acetyl-D- galactosamine (GalNAc), multivalent GalNAc, D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, glucose, multivalent glucose, multivalent fucose, glycosylated polyaminoacids, or lectins.
  • the carbohydrate-based ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker, such as:
  • the carbohydrate-based ligand is conjugated to the 3'-end of Z 11 or Z 12 , or an internal position of Z 11 or Z 12 .
  • the carbohydrate-based ligand is conjugated to the 3'-end of Z 12 .
  • one or more targeting ligands are conjugated to an internal position of Z 11 , excluding position 2 or 14.
  • the phosphate mimic modification at the 5' -end of a nucleotide sequence for the single-stranded oligonucleotide of formula (I), formula (II) (or Ila), or formula III (or Illa), can be 5'-end phosphorothioate (5'-PS), 5'-end phosphorodithioate (5'-PS2), 5' end vinylphosphonate (5' -VP), 5'-end methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl.
  • 5'-PS phosphorothioate
  • 5'-PS2 5'-end phosphorodithioate
  • 5'-VP 5' end vinylphosphonate
  • MePhos 5'-end methylphosphonate
  • the phosphate mimic is a 5 '-vinylphosphonate (VP).
  • the 5'-VP can be either 5'-E-VP isomer (i.e., trans-vinylphosphate), 5'-Z-VP isomer (i.e., cis-vinylphosphate), or mixtures thereof.
  • the phosphate mimic is a 5' -vinyl phosphonate (VP). In one embodiment, the phosphate mimic is a 5'-cyclopropyl phosphonate. In one embodiment, the phosphate mimic is a 5'-vinyl phosphate.
  • VP 5' -vinyl phosphonate
  • the phosphate mimic is a 5'-cyclopropyl phosphonate. In one embodiment, the phosphate mimic is a 5'-vinyl phosphate.
  • the single-stranded oligonucleotide further includes a phosphate or phosphate mimic at the 5'-end of the antisense strand (i.e., Z 1 ).
  • the phosphate mimic is a 5'-vinyl phosphonate (VP).
  • the phosphate mimic is a 5'-vinyl phosphonate (VP)
  • the 5' -terminal nucleotide may have the following structure, , wherein :
  • X is O or S
  • R is hydrogen, hydroxy, fluoro, or C 1-20 alkoxy (e.g., methoxy or n-hexadecyloxy);
  • R 5 C(H)-P(O)(OH) 2 and the double bond between the C5' carbon and R 5 is in the E or Z orientation (e.g., E orientation); and B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil.
  • R 5 C(H)-P(O)(OH) 2 and the double bond between the C5' carbon and R5' is in the E orientation.
  • the -CH 2 OH group at the 4 '-position of the 5 -t'erminal nucleotide is replaced with a phosphate mimic of the formula -O-CH 2 -P(O)(OR) 2 , wherein each R is independently hydrogen or C 1 -4 alkyl (e.g., one R group is hydrogen and one R group is methyl; or both R groups are hydrogen).
  • the phosphate mimic is a 5 -'cyclopropyl phosphonate (VP)
  • the 5'-end phosphate mimic is , or a salt (e.g., sodium salt) thereof, wherein B is an optionally modified nucleobase (e.g., U).
  • the 5'-end phosphate mimic is part of a modified 5'- terminal nucleotide.
  • the phosphate mimic may be part of a modified 5'- terminal nucleotide having the structure wherein B is an optionally modified nucleobase.
  • the 5'-end phosphate mimic can also include a 5'- phosphate prodrug or 5 '-phosphonate prodrug.
  • the 5 '-phosphate prodrug or 5 '-phosphonate prodrug has a structure of formulas disclosed in WO2022/147214, which is incorporated herein by reference.
  • the 5'- phosphate prodrug or 5' -phosphonate prodrug is: Pmmds ( , ((4SR,5SR)-3,3,5- trimethyl-1 ,2-dithiolan-4-ol) phosphodiester); cPmmds ( , ((4SR,5RS)-3,3,5- trimethyl- 1,2-dithiolan-4-ol) phosphodiester (Cis Pmmds)); PdArls ( ((4SR,5RS)-5-phenyl-3,3-dimethyl-1,2-dithiolan-4-ol) phosphodiester); PdAr3s ( , ((4SR,5RS)-5-(4-methylphenyl)-3,3-dimethyl-l,2-dithiolan-4-ol) phosphodiester); PdAr5s ( , ((4SR,5RS)-5-(4-methoxyphenyl)-3,3-dimethyl
  • PdAr6s ( ); Pmmd/Pmmds ( ); Pmds (
  • the 5' -phosphate prodrug or 5'-phosphonate prodrug is:
  • the siRNA containing one of the above list of 5' modified phosphate prodrugs generally has an activity comparable to that of the siRNA containing 5'-VP.
  • the 5'-phosphate prodrug or 5 '-phosphonate prodrug is:
  • the siRNA containing one of the above list of 5' modified phosphate prodrugs generally has an improved stability than that of the siRNA containing 5 '-VP and has a better or comparable activity than that of the siRNA containing 5 '-VP.
  • Another aspect of the invention relates to an oligonucleotide construct comprising two single-stranded oligonucleotides of formula (I) as described above, wherein the two single- stranded oligonucleotides are covalently bonded.
  • Another aspect of the invention relates to an oligonucleotide construct comprising two single-stranded oligonucleotides of formula (II) (or Ila) or (III) (or Illa) as described above, wherein the two single-stranded oligonucleotides are covalently bonded.
  • At least one of the single-stranded oligonucleotides forming the oligonucleotide construct is one from formula (I). In some embodiments, at least one of the single-stranded oligonucleotides forming the oligonucleotide construct is one from formula (II) (or Ila). In some embodiments, at least one of the single-stranded oligonucleotides forming the oligonucleotide construct is one from formula (III) (or Illa).
  • the covalent bonding of the two single-stranded oligonucleotides occurs at the linking group L for each single-stranded oligonucleotide.
  • the two single-stranded oligonucleotides are covalently bonded via a tethering group selected from the group consisting of oxime, aminooxy, a triazole or fused triazole, phosphodiester, phosphotriester, hydrogen phosphonate, alkyl or aryl phosphonate, phosphoramidate, phosphorothioate, a nitrogen-modified phosphorous- containing linkage (PN-linkage), methylenemethylimino, thiodiester, thionocarbamate, N,N'- dimethylhydrazine, phosphoroselenate, borano phosphate, borano phosphate ester, amide, hydroxylamino, siloxane, dialkylsiloxan
  • the tethering group is oxime, aminooxy, or a triazole or fused triazole.
  • exemplary tethering groups and exemplary process for covalently bonding two single-stranded oligonucleotides to form an oligonucleotide construct are shown in Schemes 7.1-7.4 below.
  • the two single-stranded oligonucleotides may be the same or different.
  • the two single-stranded oligonucleotides forming the oligonucleotide construct are the same.
  • the single-stranded oligonucleotide forming the oligonucleotide construct is one from formula (I).
  • the single-stranded oligonucleotide forming the oligonucleotide construct is one from formula (II) (or Ila).
  • the single-stranded oligonucleotide forming the oligonucleotide construct is one from formula (III) (or Illa).
  • the two single-stranded oligonucleotides forming the oligonucleotide construct are different.
  • the two single-stranded oligonucleotides forming the oligonucleotide construct are two different single-stranded oligonucleotides from formula (I).
  • Z 1 and/or Z 2 of one single-stranded oligonucleotide contains different modifications than Z 1 and/or Z 2 of the other single-stranded oligonucleotide.
  • L of one single-stranded oligonucleotide is different than L of the other single- stranded oligonucleotide.
  • Q 1 and/or Q 2 of one single-stranded oligonucleotide is different than Q 1 and/or Q 2 of the other single-stranded oligonucleotide.
  • one single-stranded oligonucleotide contains a ligand that is different than the other single-stranded oligonucleotide. For instance, one single-stranded oligonucleotide contains a ligand, and the other single-stranded oligonucleotide does not contain a ligand or contains a different ligand. In some embodiments, one single-stranded oligonucleotide contains a ligand that is at a different location than the ligand on the other single- stranded oligonucleotide.
  • the two single-stranded oligonucleotides forming the oligonucleotide construct are two different single-stranded oligonucleotides from formula (II) (or Ila) or (III)(or Illa).
  • one single-stranded oligonucleotide is from formula (II) (or Ila)
  • the other single-stranded oligonucleotide is from formula (III)(or Illa).
  • Z 11 and/or Z 12 of one single-stranded oligonucleotide contains different modifications than Z 11 and/or Z 12 of the other single-stranded oligonucleotide.
  • L of one single-stranded oligonucleotide is different than L of the other single- stranded oligonucleotide.
  • one single-stranded oligonucleotide contains a L, and the other single-stranded oligonucleotide does not contain a L or contains a different L.
  • Q s of one single-stranded oligonucleotide is different than Q s of the other single-stranded oligonucleotide.
  • one single-stranded oligonucleotide contains a Q s
  • the other single-stranded oligonucleotide does not contain a Q s or contains a different Q s
  • one single-stranded oligonucleotide contains a ligand that is different than the other single-stranded oligonucleotide.
  • one single- stranded oligonucleotide contains a ligand, and the other single-stranded oligonucleotide does not contain a ligand or contains a different ligand.
  • one single-stranded oligonucleotide contains a ligand that is at a different location than the ligand on the other single- stranded oligonucleotide.
  • the two single-stranded oligonucleotides forming the oligonucleotide construct are two different single-stranded oligonucleotides, having one single- stranded oligonucleotide from formula (I) and another single-stranded oligonucleotide from formula (II) (or Ila) or (III)(or Illa).
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the single-stranded oligonucleotide described above according to formula (I), and a pharmaceutically acceptable excipient.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the single-stranded oligonucleotide described above according to formula (II) (or (Ila)) or (III) (or (Illa)), and a pharmaceutically acceptable excipient.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the oligonucleotide construct described above, comprising two single-stranded oligonucleotides according to formula (I), (II) (or (Ila)), or (III) (or (Illa)), and a pharmaceutically acceptable excipient.
  • Another aspect of the invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising contacting the cell of the subject with, or administering to the subject, the single-stranded oligonucleotide described above according to formula (I), in an amount sufficient to inhibit the activity or expression of the one or more target genes in the cell of the subject.
  • Another aspect of the invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising contacting the cell of the subject with, or administering to the subject, the single-stranded oligonucleotide described above according to formula (II) (or (Ila)) or (III) (or (Illa)), in an amount sufficient to inhibit the activity or expression of the one or more target genes in the cell of the subject.
  • Another aspect of the invention relates to a method for inhibiting the expression of one or more target genes in a subject, comprising contacting the cell of the subject with, or administering to the subject, the oligonucleotide construct described above, comprising two single- stranded oligonucleotides according to formula (I), (II) (or (Ila)), or (III) (or (Illa)), in an amount sufficient to inhibit the activity or expression of the one or more target genes in the cell of the subject.
  • the cell is within a subject.
  • the subject is a human.
  • the subject is a non-human mammal, e.g., a rhesus monkey, a cynomolgous monkey, a mouse, or a rat.
  • the single-stranded oligonucleotide is capable of inhibiting the activity or expression of the one or more target genes in a tissue of the subject by at least 15% each relative to an appropriate control (e.g., as compared to an untreated or placebo-treated subject, or as compared to a reference value, including, e.g., target mRNA or protein levels in the treated subject measured before the treatment with the single- stranded oligonucleotide or the double-stranded nucleic acid agent occurred), optionally by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% each relative to an appropriate control.
  • the appropriate control is an untreated subject.
  • the appropriate control is a reference value, e.g.,
  • Figure 1 A is a scheme showing the sequence design of the exemplary single- stranded oligonucleotides, and their conjugations to the GalNAc ligands.
  • Figure IB is a scheme showing the sequence design of the exemplary single-stranded oligonucleotides, as shown in Figure 1 A, but in the form of a loopmer having a loop and an intra-strand duplexed region between the corresponding antisense and sense nucleotides.
  • Figure 2 shows the loss of full-length for the exemplary single-stranded oligonucleotides and strands of the parent siRNA duplex listed in Tables 1 and 3, after incubation of the oligonucleotides in plasma.
  • Figure 3 is a scheme showing the plasma metabolism summary for the exemplary single- stranded oligonucleotides listed in Table 1.
  • Figure 4 is a scheme showing the liver homogenate metabolism summary for the exemplary single-stranded oligonucleotides and the parent siRNA duplex listed in Tables 1 and 3.
  • Figure 5A-C illustrates the gene silencing effects of the exemplary single-stranded loop oligonucleotides compared against the parent siRNAs in mice.
  • GalNAc-siRNAs with double strandard sense and antisense strands On-1
  • GalNAc- single-stranded loop oligonucleotides On-2 to On-11).
  • Figure 6 shows the total ion chromatograms illustrating the major metabolites of parent siRNA or the exemplary single- stranded loop oligonucleotides (a. On-1 b. On-2 c. On- 3 d. On-5 e. On-6 f. On-7 g. On-8 h. On-9 i. On- 10 j. On-11) formed at 24 hours in rat plasma.
  • Figure 7 shows the total ion chromatograms illustrating the major metabolites of parent siRNA or the exemplary single- stranded loop oligonucleotides (a. On-1 b. On-2 c. On- 3 d. On-5 e. On-6 f. On-7 g. On-8 h. On-9 i. On- 10 j. On-11) formed at 24 hours in rat liver homogenate.
  • Figures 8A and 8B show the intesnity of parent loopmerRNA/formation of 22- 23mer antisense metabolite correlates with %mTTR knockdown.
  • Figure 9A shows the loss of intensity of parent RNA from in vitro liver incubations correlated with %mTTR knockdown in vivo. An intenisty of zero is used for loopmers where full length loopmerRNA was not identified.
  • Figure 9B shows the formation of 22/23 antisense for each single- stranded loop oligonucleotide from in vitro liver incubations correlated with %mTTR knockdown in vivo.
  • Figure 9 shows the results of knockdown of TTR mRNA with and without VP, comparing On-2, On-3, On-9, and On-10.
  • Figure 10 is a scheme showing the sequence design of exemplary single-stranded oligonucleotides in the form of a loopmer having a loop and an intra-strand duplexed region between the corresponding antisense and sense nucleotides, and their conjugations to the GalNAc ligands, as compared to the parent siRNA duplex.
  • Various loop design and various chemistries for the single-stranded oligonucleotides in connection with their stability in liver homogenate and plasma are illustrated in the figure.
  • the single-stranded oligonucleotides having a loop region containing a 3-nt loop was stable in liver homogenate for 24 hours and in plasma for 8 hours.
  • the single-stranded oligonucleotides having a loop region containing a 7-nt loop was semi-labile in liver homogenate for 24 hours and in plasma for 8 hours.
  • the single-stranded oligonucleotides having a loop region containing a 7-nt loop was labile in liver homogenate for 24 hours and in plasma for 8 hours.
  • Figures 11A-11B show the results of the metabolism and in vivo knockdown of the exemplary single-stranded oligonucleotides listed in Figure 10.
  • Figure 11A shows the inhibition of mTTR expression by the exemplary single-stranded oligonucleotides (listed in Figure 10) in a mouse at a single dosage of 0.2 mg/kg.
  • the label for “stable loop'' corresponds to A-1700636 shown in Figure 10; the label for “semi-labile loop'' corresponds to A-492540 in Figure 10; the label for “labile loop'' corresponds to A-511271 in Figure 10; the label for “canonical duplex'' corresponds to the parent siRNA AD-64228.
  • Figure 11B shows the inhibition of mTTR expression by certain exemplary single-stranded oligonucleotides (listed in Figure 10) in a mouse at a single dosage of 0.2 mg/kg, as compared to the same oligonucleotide but with a 5'-(E)-vinylphosphonate (VP) modification.
  • the label “semi-labile loop + 5'-VP'' refers to the sequence of A-492540 (“semi-labile loop'') but with a 5'-(E)-VP modification.
  • canonical duplex + 5' -VP'' refers to the sequence of parent siRNA AD-64228 (“canonical duplex'') but with a 5'-(E)-VP modification.
  • Figure 12 show the results of in vivo knockdown of the exemplary single-stranded oligonucleotides as compared to parent duplexes and controls (Table 7), in a mouse at a single dosage of 2.5 mg/ml.
  • the samples in the gragh from left to right are PBS, AD- 579804, A-4102742, A-3903365, A-3903366, AD-1953663, A-3903367, A-3903368, AD- 1983263, AD-1983265, respectively.
  • the inventors have designed a novel strategy to prepare a single-stranded loop oligonucleotide using two chemically modified oligonucleotides capable of forming an intra- strand duplexed region and connecting the two oligonucleotides by a cleavable linking group, generating a single-stranded construct.
  • the single-stranded loop oligonucleotide is designed to cleave at a suitable rate for the single-stranded construct to be cleaved into a double- stranded RNAi agent that is effective in vivo.
  • the single-stranded loop oligonucleotide are synthesized as single strands and self-anneal due to sequence complementarity and are purified as single strands. Delivery ligands such as triantennary GalNAc can be readily incorporated during synthesis.
  • the single-stranded loop oligonucleotide described herein has the stability in plasma with the ability to metabolize and release siRNAs efficiently in vivo.
  • the single-stranded loop oligonucleotide discussed herein provides an improved design to simplify the manufacture and purification of RNAi agent by increasing the throughput and reducing the overall synthesis time, yet at the same time preserving or improving the efficacy of the RNAi agent when being cleaved in vivo.
  • One aspect of the invention relates to a single-stranded oligonucleotide capable of inhibiting the expression of a target gene, having a sequence represented by formula (I): (5' - Z 1 - 3')-Q 1 L-Q 2 -(5' - Z 2 - 3')
  • Z 1 is a first oligonucleotide, comprising 15 - 100 optionally modified nucleotides that is substantially complementary to a target gene;
  • Z 2 is a second oligonucleotide, comprising 15 - 100 optionally modified nucleotides that is substantially complementary to Z 1 ;
  • Z 1 and Z 2 are capable of forming an intra-strand duplexed region comprising 3 or more consecutive base pairs;
  • L is a linking group
  • Q 1 and Q 2 each independently represent 0 to 12 optionally modified nucleotides; and at least one nucleotide in formula (I) is a modified nucleotide.
  • the single-stranded oligonucleotide is formed by connecting the two oligonucleotides by a linking group L.
  • Some exemplary single-stranded oligonucleotide constructs are illustrated in Schemes 1 and 2.
  • Z 1 represents a first oligonucleotide that is substantially complementary to a target gene (e.g., an antisense strand); and Z 2 represents is a second oligonucleotide that is substantially complementary to Z 1 (e.g., a sense strand).
  • Z 1 and Z 2 can form an intra-strand duplexed region between the corresponding nucleotides of Z 1 and Z 2 , and the single-stranded oligonucleotide contains a loop region formed by the linking group L (and possibly Q 1 and Q 2 ).
  • Q 1 and Q 2 each independently can be absent.
  • Q 1 and Q 2 each independently can be present as an overhang to the first oligonucleotide Z 1 and the second oligonucleotide Z 2 , respectively.
  • L is a linking group that can contain modified or unmodified nucleotides.
  • L can contain non-nucleotide based linkers, such as Q304.
  • the nucleotides of the entire single-strand oligonucleotide can contain various chemical modifications such as DNA, RNA, 2'-F, or 2'-OMe .
  • the single-strand oligonucleotide further comprises a ligand, e.g., 3 GalNAc derivatives attached through a trivalent branched linker, at the 3' end of Z 2 (e.g., a sense strand).
  • the single-strand oligonucleotide further comprises a phosphate or phosphate mimic (e.g., 5' end vinylphosphonate (5'-VP)) at the 5'-end of Z 1 (e.g., an antisense strand).
  • Each of the first oligonucleotide Z 1 and second oligonucleotide Z 2 can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • Each of the first oligonucleotide Z 1 and second oligonucleotide Z 2 may have about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, about 10 to about 20 nucleotides, about 15 to about 50 nucleotides, about 15 to about 40 nucleotides, about 15 to about 35 nucleotides, about 15 to about 30 nucleotides, about 15 to about 25 nucleotides, about 15 to about 20 nucleotides, or about 18 to about 20 nucleotides in length.
  • each of the first oligonucleotide Z 1 and second oligonucleotide Z 2 is at least 15 nucleotides in length. In one embodiment, each of the first oligonucleotide Z 1 and second oligonucleotide Z 2 is at least 18 nucleotides in length.
  • Another aspect of the invention relates to a single-stranded oligonucleotide according to formula (II) or (III):
  • Z 11 is a first oligonucleotide, comprising 15 - 100 optionally modified nucleotides that is substantially complementary to a target gene;
  • Z 12 is a second oligonucleotide, comprising 10 - 100 optionally modified nucleotides that is substantially complementary to Z 11 ;
  • Z 11 and Z 12 are capable of forming an intra-strand duplexed region comprising 7 or more consecutive base pairs;
  • Q s represents 0 to 12 optionally modified nucleotides
  • L is an optional linking group; at least one nucleotide in formula (II) is a modified nucleotide; and at least one nucleotide in formula (III) is a modified nucleotide, wherein at least one nucleotide at the 3' end of Z 11 , for formula (II), at least one nucleotide at the 5' end of Z 11 , for formula (III), in either case together with L and Q s form a loop region connecting Z 11 and Z 12 .
  • the single-stranded oligonucleotide is formed by connecting the two oligonucleotides, optionally by a linking group L.
  • the single-stranded oligonucleotide is represented by formula (Ila) or formula (Illa): , wherein:
  • Z 11 comprises W — LP, W forms an intra-strand duplexed region at least 7 base pairs with Z 12 ,
  • LP optionally together with L, forms a loop between W and Z 12 at the 3' -end or 5'- end, represents an optional presence of L
  • > represents an optional overhang at 5'-end or 3'-end of Z 11 .
  • > represents an optional overhang at 5' -end or 3' -end of Z 12 .
  • Some exemplary single-stranded oligonucleotides may have the orientation (e.g., 5'-3' orientation) and connections of Z 11 and Z 12 , as defined in formula (II) or (Ila), as illustrated by Schemes 1B.1- 1B.5.
  • the PS intemucleotide linkages illustrated in each of Schemes 1B.1- 1B.5 are exemplary and may be present or absent.
  • the illustrated PS internucleotide linkages at the 3' - and 5'-ends are present, while the internal PS internucleotide linkages are absent.
  • Z 11 represents a first oligonucleotide that is substantially complementary to a target gene (e.g., an antisense strand); and Z 12 represents is a second oligonucleotide that is substantially complementary to Z 11 (e.g., a sense strand).
  • Z 11 and Z 12 can form an intra-strand duplexed region between the corresponding nucleotides of Z 11 and Z 12 , and the single- stranded oligonucleotide contains a loop region LP (possibly including a linking group L; not marked).
  • Q s can be absent.
  • Q s is represented by a and b, which may be spacers and can be any optionally modified nucleotide that form matched or mismatched base pairs with their opposite nucleotides in Z 11 (e.g., the two corresponding nucleotides at positions 17 and 18, in Scheme 1B.1).
  • both a and b form matched base pairs with their opposite nucleotides in Z 11 .
  • both a and b form mismatched base pairs with their opposite nucleotides in Z 11 .
  • one of a and b forms a matched base pair with its opposite nucleotide in Z 11 ; and another of a and b forms a mismatched base pair with its opposite nucleotide in Z 11 .
  • b forms a mismatched base pair with its opposite nucleotide in Z 11 (e.g., b is mismatched to the nucleotide at position 17, as shown in Scheme 1B.1).
  • a forms a mismatched base pair with its opposite nucleotide in Z 11 (e.g., a is mismatched to the nucleotide at position 18, as shown in Scheme 1B.1).
  • the single-stranded oligonucleotide contains one or two phosphorothioate internucleotide linkage modifications (e.g., two consecutive phosphorothioate internucleotide linkage modifications) within first 6 nucleotides or last 6 nucleotides of Z 11 or Z 12 (i.e., one or two phosphorothioate intemucleotide linkage modifications between nucleotides at terminal 6 positions from either the 5' end or the 3' end for either Z 11 or Z 12 ).
  • one or two phosphorothioate internucleotide linkage modifications e.g., two consecutive phosphorothioate internucleotide linkage modifications
  • the single-stranded oligonucleotide contains two consecutive phosphorothioate intemucleotide linkage modifications within first 3 nucleotides or last 3 nucleotides of Z 11 or Z 12 (i.e., the intemucleotide linkages between terminal 3 positions from either the 5' end or the 3' end for either Z 11 or Z 12 are modified with wo consecutive phosphorothioate intemucleotide linkage modifications, e.g., the phosphorothioate intemucleotide linkage modifications shown as “stars'' in iii) of Scheme 1B.1).
  • the single-stranded oligonucleotide contains one or two phosphorothioate intemucleotide linkage modifications (e.g., two consecutive phosphorothioate intemucleotide linkage modifications) within last 8 nucleotides of Z 11 .
  • the single-stranded oligonucleotide has Z 11 of 23 nucleotides in length, and contains two phosphorothioate intemucleotide linkage modifications between nucleotides at positions 16-23 (e.g., two phosphorothioate intemucleotide linkage modifications between nucleotides at positions 16-17, 17-18, 18-19, 19-20, 20-21, 21-22 ofZ 11 , as shown in Scheme 1B.1).
  • the single-stranded oligonucleotide contains one or two phosphorothioate internucleotide linkage modifications (e.g., two consecutive phosphorothioate internucleotide linkage modifications) within first 3 nucleotides of Z 12 (e.g., one or two phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and/or 2-3 of Z 12 , as shown in Scheme 1B.1).
  • the single- stranded oligonucleotide contains two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 , two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides (e.g., at positions 14-15 and 15-16) of Z 12 , and two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 , as shown in ii) of Scheme 1B.1.
  • the single-stranded oligonucleotide can comprise a 5 '-phosphate or 5-phosphate mimic modification, as described herein, at the 5'-end of a nucleotide sequence (e.g., Z 11 and/or Z 12 ) (e.g., a 5'-end vinylphosphonate (5'-VP), at the 5'-end of Z 11 , as shown in ii) of Scheme 1B.1).
  • a nucleotide sequence e.g., Z 11 and/or Z 12
  • 5'-VP 5'-end vinylphosphonate
  • the single-stranded oligonucleotide may further comprise one or more ligands (e.g., a lipophilic moiety for extrahepatic delivery, as described herein).
  • one or more lipophilic moieties are conjugated independently to one or more of the internal positions (i.e., non-terminal positions) of Z 11 .
  • one or more lipophilic moieties are conjugated independently to one or more of positions 11, 12, 13 from 5'-end of Z 11 , as shown in ii) of Scheme 1B.1.
  • one or more lipophilic moieties are conjugated independently to one or more of the internal positions of Z 11 , excluding position 2 or 14.
  • one or more lipophilic moieties are conjugated independently to one or more positions of Z 12 and/or Q s , excluding the positions of Q s and/or Z 12 paired to positions 11, 12, and 13 from the 5'-end of Z 11 , as shown in ii) of Scheme 1B.1.
  • the single-stranded oligonucleotide may further comprise one or more targeting ligands (e.g., liver targeting carbohydrate-based ligand, as described herein).
  • one or more targeting ligands e.g., carbohydrate-based ligands
  • the 3'-end of Z 11 or Z 12 as shown in iii) of Scheme IB .1
  • one or more targeting ligands are conjugated to an internal position of Z 11 , excluding position 2 or 14.
  • one or more targeting ligands are conjugated to the 3' -end of Z 12 , as shown in iii) of Scheme 1B.1.
  • the single-stranded oligonucleotide when the single-stranded oligonucleotide contains a terminal conjugation of a ligand to the 5'-end or 3' -end nucleotide, or contains a terminal conjugation of an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide to the 5'-end or 3' -end nucleotide, then at that terminus, the above intemucleotide linkage modifications to the terminal nucleotide can be omitted (e.g., one or two phosphorothioate intemucleotide linkage modifications between nucleotides at terminal 6 or 3 positions to the 3'-end of Z 12 can be omitted, due to the conjugation of a ligand to the 3'-end of Z 12 , as shown in iii) of Scheme 1B.1).
  • Z 11 comprises 19 - 23 optionally modified nucleotides
  • Z 12 comprises 12 - 16 optionally modified nucleotides
  • Q s comprises 2 optionally modified nucleotides, as shown in Scheme 1B.2.
  • the duplexed region formed by Z 11 and Z 12 at the non-loop terminal (e.g., the 5'-end of Z 11 ) has a blunt end, as shown in Scheme 1B.2.
  • Z 11 comprises 19 - 23 optionally modified nucleotides
  • Z 12 comprises 12 - 16 optionally modified nucleotides
  • Q s comprises 2 optionally modified nucleotides.
  • the 3-5 terminal nucleotides of Z 11 contain modifications selected from the group consisting of 2' -deoxy nucleotide (dN), a 2'-deoxy-2'-fluoronucleotide (fN), a ribonucleotide (rN), 2'-O- methylnucleotide (mN), and 2'-aranucleotide (aN), to encourage cleavage.
  • the 5 terminal nucleotides of Z 11 , connected to L, Q s , or Z 12 have modifications selected from the group consisting of
  • #-mN-mN-rN-rN-rN-** and wherein: # is the bond to Z 11 and ** is the bond to L, Q s , or Z 12 , dN represents a 2' -deoxy nucleotide, fN represents a 2 '-deoxy-2' -fluoro nucleotide, rN represents a ribonucleotide, and mN represents a 2'-O-methyl nucleotide.
  • the 3 terminal nucleotides of Z 11 connected to L, Q s , or Z 12 , have modifications independently selected from the group consisting of 2'-fluoro, 2'-deoxy, and 2'-OH, such as:
  • the single-stranded oligonucleotide contains two consecutive phosphorothioate internucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 , and two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides of Z 12 , as shown in Scheme 1B.3.
  • the single-stranded oligonucleotide contains six terminal phosphorothioate internucleotide linkages modifications: two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 , two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides of Z 12 , and two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 , as shown in Scheme 1B.3.
  • the second oligonucleotide Z 12 optionally together with Q s , contains at least one motif of three consecutive 2'-F modifications, and the nucleotide next to the motif is not 2'-F modified.
  • the position of the motif of three consesutive modifications is characterized by one or the followings: the motif is at Q s , positions 1 and 2 of Z 12 , optionally Z 11 is 19 nucleotides in length; the motif is at positions 1, 2, and 3 of Z 12 , optionally Z 11 is 20 nucleotides in length; the motif is at positions 2, 3, and 4 of Z 12 , optionally Z 11 is 21 nucleotides in length; the motif is at positions 3, 4, and 5 of Z 12 , optionally Z 11 is 22 nucleotides in length; or the motif is at positions 4, 5, and 6 of Z 12 , optionally Z 11 is 23 nucleotides in length.
  • the first oligonucleotide Z 11 contains a modification that is not 2'-O-methyl at positions 2 and 14. In one embodiment, as shown in Scheme 1B.3, the first oligonucleotide Z 11 contains a 2'-F modification at position 14.
  • the first oligonucleotide Z 11 contains one or more 2'-deoxy (DNA) modifications at positions 2, 5, 7, and 12.
  • Z 11 comprises 19 - 23 optionally modified nucleotides
  • Z 12 comprises 12 - 16 optionally modified nucleotides
  • Q s comprises 2 optionally modified nucleotides.
  • the 3-5 terminal nucleotides of Z 11 contain modifications selected from the group consisting of 2' -deoxy nucleotide (dN), a 2'-deoxy-2'-fluoronucleotide (fN), a ribonucleotide (rN), 2'-O- methylnucleotide (mN), and 2'-aranucleotide (aN), to encourage cleavage.
  • the 5 terminal nucleotides of Z 11 , connected to L, Q s , or Z 12 have modifications selected from the group consisting of:
  • # is the bond to Z 11 and ** is the bond to L, Q s , or Z 12 , dN represents a 2' -deoxy nucleotide, fN represents a 2' -deoxy-2' -fluoro nucleotide, rN represents a ribonucleotide, and mN represents a 2'-O-methyl nucleotide.
  • the 3 terminal nucleotides of Z 11 connected to L, Q s , or Z 12 , have modifications independently selected from the group consisting of 2'-fluoro, 2'-deoxy, and 2'-OH, such as:
  • the single-stranded oligonucleotide contains two consecutive phosphorothioate internucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 , and two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides of Z 12 , as shown in Scheme 1B.4.
  • the single-stranded oligonucleotide contains six terminal phosphorothioate internucleotide linkages modifications: two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 , two consecutive phosphorothioate internucleotide linkage modifications between last 3 nucleotides of Z 12 , and two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 , as shown in Scheme 1B.4.
  • the second oligonucleotide Z 12 optionally together with Q s , contains at least one motif of three consecutive 2'-F modifications, and the nucleotide next to the motif is not 2'-F modified.
  • the position of the motif of three consesutive modifications is characterized by one or the followings: the motif is at Q s , positions 1 and 2 of Z 12 , optionally Z 11 is 19 nucleotides in length; the motif is at positions 1, 2, and 3 of Z 12 , optionally Z 11 is 20 nucleotides in length; the motif is at positions 2, 3, and 4 of Z 12 , optionally Z 11 is 21 nucleotides in length; the motif is at positions 3, 4, and 5 of Z 12 , optionally Z 11 is 22 nucleotides in length; or the motif is at positions 4, 5, and 6 of Z 12 , optionally Z 11 is 23 nucleotides in length.
  • Z 12 optionally together with Q s , contains a 2'-O-methyl or 2'-F modification at a position that is 2 positions before the motif of three consecutive 2'-F modifications (position n-2, if the motif starts at position n), provided that the position is not part of Z 11 .
  • Z 12 optionally together with Q s , contains a 2'-F modification at a position that is 2 positions before the motif of three consecutive 2'-F modifications (position n-2, if the motif starts at position n), provided that the position is not part of Z 11 .
  • the first oligonucleotide Z 11 contains a modification that is not 2'-O-methyl at positions 2 and 14. In one embodiment, as shown in Scheme 1B.4, the first oligonucleotide Z 11 contains a 2'-F modification at position 14.
  • the first oligonucleotide Z 11 contains one or more 2'- F modifications at positions 2, 6, 8, 9, 14, and 16.
  • L is present in formula (II) (or Ila) or formula III (or Illa), and contains a linking moiety represented by a formula: #-(N) n -**.
  • # is the bond to Z 11 and ** is the bond to Q s or Z 12 ;
  • n is 3 to 12; and each N is independently a linking monomer having a chain length of 3 or more atoms.
  • n is 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5. In one embodiment, n is 3.
  • all the linking monomer of L (e.g., Q304), together with LP form a loop between W (Z 11 ) and Z 12 .
  • one or more of the linking monomers of L (e.g., Q304), together with LP forms a loop between W (Z 11 ) and Z 12 , and one or more of the linking monomers of L (e.g., Q304) is not in the loop region.
  • one or more of the linking monomers of L (e.g., Q304), together with LP forms a loop between W (Z 11 ) and Z 12 , and one or more of the linking monomers of L (e.g., Q304) is not in the loop and is connected to Q s (a).
  • one or more of the linking monomers of L (e.g., Q304), together with LP forms a loop between W (Z 11 ) and Z 12 , and one or more of the linking monomers of L (e.g., Q304) is not in the loop and is connected to Z 12 .
  • one or more linking moieties (N) in L may be an optionally modified nucleotide.
  • one or more linking moieties (N) in L may be independently selected from the group consisting of a 2' -deoxynucleotide (dN), a 2' -deoxy - 2'-fluoro nucleotide (fN), a ribonucleotide (rN), 2 '-O-m ethylnucleotide (mN), and 2'-ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2 -'ara ribonucleotide).
  • dN 2' -deoxynucleotide
  • fN 2' -deoxy - 2'-fluoro nucleotide
  • rN a ribonucleotide
  • mN 2'
  • one or more linking moieties (N) in L may be independently selected from the group consisting of Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, QI 1, Q150, Q151, Q173, Q221, Q222, Q367, and Q368.
  • L contains a triplet of Q304.
  • the single-stranded oligonucleotide contains two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 , two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides of Z 12 , and two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 , as shown in Scheme 1B.4.
  • the single-stranded oligonucleotide contains one or two phosphorothioate intemucleotide linkage modifications (e.g., two consecutive phosphorothioate intemucleotide linkage modifications) within last 5, 6, or 7 nucleotides of Z 11 , as shown in Scheme 1B.5.
  • the single-stranded oligonucleotide contains six terminal phosphorothioate intemucleotide linkages modifications: two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 , two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides of Z 12 , and two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 , as shown in Scheme 1B.5.
  • the single-stranded oligonucleotide contains eight terminal phosphorothioate intemucleotide linkages modifications: two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 11 ; two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides of Z 12 ; two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 ; and two consecutive phosphorothioate intemucleotide linkage modifications) within last 5, 6, or 7 nucleotides of Z 11 , as shown in Scheme 1B.5.
  • Some exemplary single-stranded oligonucleotides may have the orientation (e.g., 5'-3' orientation) and connections of Z 11 and Z 12 , as defined in formula (III) or (Illa), as illustrated by Schemes 2B.1- 2B.4.
  • Z 11 comprises 19 - 23 optionally modified nucleotides
  • Z 12 comprises 16 - 19 optionally modified nucleotides
  • Q s may be absent or present comprising 2 optionally modified nucleotides.
  • Z 11 comprises 23 optionally modified nucleotides
  • Z 12 comprises 16-19 optionally modified nucleotides.
  • Z 11 comprises 21 optionally modified nucleotides
  • Z 12 comprises 14-17 optionally modified nucleotides.
  • Z 11 comprises 23 optionally modified nucleotides
  • Z 12 comprises 18-21 optionally modified nucleotides.
  • Z 11 comprises 21 optionally modified nucleotides
  • Z 12 comprises 16-19 optionally modified nucleotides.
  • the duplexed region formed by Z 11 and Z 12 at the non-loop terminal (e.g., the 3 '-end of Z 11 ) has a blunt end, as shown in Schemes 2B.3 and 2B.4.
  • Z 11 at the non-loop terminal has an overhang of 1-3 nucleotides in length. In one embodiment, Z 11 at the non-loop terminal has an overhang of 2 nucleotides in length (e.g., at the 3'-end of Z 11 , as shown in Schemes 2B.1 and 2B.2). In one embodiment, Z 11 at the non-loop terminal has an overhang of 2 nucleotides in length and has a phosphorothioate intemucleotide linkage between the two overhang nucleotides, as shown in Schemes 2B.1 and 2B.2.
  • Z 11 at the non-loop terminal has an overhang of 2 nucleotides in length (e.g., at the 3'-end of Z 11 ) and has two phosphorothioate intemucleotide linkages between the terminal 3 nucleotides (e.g., at the 3'-end of Z 11 ), in which 2 of the 3 nucleotides are the overhang nucleotides, and the third is the paired nucleotide next to the overhang nucleotide, as shown in Schemes 2B.1 and 2B.2.
  • the single-stranded oligonucleotide contains one or two phosphorothioate intemucleotide linkage modifications (e.g., two consecutive phosphorothioate intemucleotide linkage modifications) within first 4 nucleotides of Z 11 or within first 3 nucleotides of Z 12 , as shown in Schemes 2B.1 and 2B.2.
  • the single-stranded oligonucleotide contains six terminal phosphorothioate intemucleotide linkages modifications: two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 ; two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides between first 4 nucleotides of Z 11 ; and two consecutive phosphorothioate intemucleotide linkage modifications between last 3 nucleotides of Z 11 , as shown in Schemes 2B.1-2B.4.
  • the single-stranded oligonucleotide contains eight terminal phosphorothioate intemucleotide linkages modifications: two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides at positions 1-2 and 2-3 of Z 12 ; two consecutive phosphorothioate internucleotide linkage modifications between last 3 nucleotides of Z 12 ; two consecutive phosphorothioate intemucleotide linkage modifications between nucleotides between first 4 nucleotides of Z 11 ; and two consecutive phosphorothioate internucleotide linkage modifications between last 3 nucleotides of Z 11 , as shown in Schemes 2B.1-2B.4.
  • the single-stranded oligonucleotide when the single-stranded oligonucleotide contains a terminal conjugation of a ligand to the 5'-end or 3' -end nucleotide, or contains a terminal conjugation of an abasic nucleotide, an inverted nucleotide, or an inverted abasic nucleotide to the 5'-end or 3' -end nucleotide, then at that terminus, the above intemucleotide linkage modifications to the terminal nucleotide can be omitted (e.g., one or two phosphorothioate intemucleotide linkage modifications between nucleotides at terminal 6 or 3 positions to the 5'-end of Z 12 can be omitted, due to the conjugation of a ligand to the 5'-end of Z 12 , as shown in Schemes 2B.1-2B.4).
  • the intra-strand duplexed region formed by Z 11 and Z 12 may contain all consecutive base pairs, or may contain up to 3 (e.g., 0, 1, 2, or 3) mismatch based pairs.
  • Z 12 may contain one nucleotide that forms mismatched base pair with the opposite nucleotidein Z 11 (e.g., the last nucleotide of Z 12 , or the n-l th nucleotide if the last nucleotide is the n th nucleotide).
  • the 3-5 terminal nucleotides of Z 11 contain modifications selected from the group consisting of 2' -deoxynucleotide (dN), a 2'-deoxy-2'-fluoronucleotide (fN), a ribonucleotide (rN), 2'-O-methylnucleotide (mN), and 2'-aranucleotide (aN), to encourage cleavage.
  • the 5 terminal nucleotides of Z 11 , connected to L, Q s , or Z 12 have modifications selected from the group consisting of
  • the 3 terminal nucleotides of Z 11 connected to L, Q s , or Z 12 , have modifications independently selected from the group consisting of 2 '-fluoro, 2 -'deoxy, and 2 -O' H, such as:
  • #-dN-dN-rN-** #-dN-rN-dN-**, #-rN-dN-dN-**, #-rN-rN-dN-**, #-rN-dN-rN-**, #-dN-rN-**, #-dN-rN-**, and #-rN-rN-rN-**.
  • L is present in formula (II) (or Ila) or formula III (or Illa), and contains a linking moiety represented by a formula: #-(N) n -**.
  • # is the bond to Z 11 and ** is the bond to Q s or Z 12 ;
  • n is 3 to 12; and each N is independently a linking monomer having a chain length of 3 or more atoms.
  • n is 3 to 8, 4 to 8, 3 to 7, 4 to 7, 3 to 6, 4 to 6, or 3 to 5.
  • n is 3.
  • all the linking monomer of L (e.g., Q304), together with LP form a loop between W (Z 11 ) and Z 12 .
  • one or more of the linking monomers of L (e.g., Q304), together with LP forms a loop between W (Z 11 ) and Z 12 , and one or more of the linking monomers of L (e.g., Q304) is not in the loop region.
  • one or more of the linking monomers of L (e.g., Q304), together with LP forms a loop between W (Z 11 ) and Z 12 , and one or more of the linking monomers of L (e.g., Q304) is not in the loop and is connected to Q s (a).
  • one or more of the linking monomers of L (e.g., Q304), together with LP forms a loop between W (Z 11 ) and Z 12 , and one or more of the linking monomers of L (e.g., Q304) is not in the loop and is connected to Z 12 .
  • one or more linking moieties (N) in L may be an optionally modified nucleotide.
  • one or more linking moieties (N) in L may be independently selected from the group consisting of a 2' -deoxynucleotide (dN), a 2' -deoxy - 2'-fluoro nucleotide (fN), a ribonucleotide (rN), 2 '-O-m ethylnucleotide (mN), and 2'-ara nucleotide (aN) (e.g., 2'-ara-2'-deoxy, 2'-ara-2'-F, 2'-ara-2'-OMe, or 2 -'ara ribonucleotide).
  • dN 2' -deoxynucleotide
  • fN 2' -deoxy - 2'-fluoro nucleotide
  • rN a ribonucleotide
  • mN 2'
  • one or more linking moieties (N) in L may be independently selected from the group consisting of Y16, Y34, Q48, Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316, Q317, Q8, QI 1, Q150, Q151, Q173, Q221, Q222, Q367, and Q368.
  • L contains a triplet of Q304.
  • the single-stranded oligonucleotide nucleotide sequence may be a substrate cleavable by DICER.
  • Another aspect of the invention relates to an oligonucleotide construct comprising two single-stranded oligonucleotides of formula (I) as described above, wherein the two single- stranded oligonucleotides are covalently bonded.
  • the two single- stranded oligonucleotides are covalently bonded via a tethering group.
  • Exemplary tethering groups and exemplary process for covalently bonding two single-stranded oligonucleotides to form an oligonucleotide construct are shown in Schemes 7.1-7.4 below.
  • Linkers/T ethers may be contained in the linking group L in the single-stranded oligonucleotide to connect the two oligonucleotides to form the single-stranded oligonucleotide.
  • Linkers/T ethers may be contained in the tethering group in the oligonucleotide construct (i.e., the gemini style) to connect the two single-stranded oligonucleotides to form the oligonucleotide construct.
  • Linkers/tethers can also be used to connect the ligand to the single-stranded oligonucleotide, e.g., via a carrier.
  • linker [0334]
  • linker "linkage,'' “linking group,'' “linking moiety,'' and “tether''” can be used interchangeably.
  • the linking group L may contain multiple linkers/tethers, each may be the same or different.
  • the linking group L in the single-stranded oligonucleotide may be a nucleotide- based or non-nucleotide-based linker.
  • the linking group L may be a stable linker that is stable in a biological fluid (e.g., in plasma or artificial cerebrospinal fluid).
  • the linking group L may be a cleavable linking group (e.g., a bio-cleavable linker).
  • Linkers/ tethers may be connected to a ligand at a “tethering attachment point (TAP).
  • TAP tethering attachment point
  • Linkers/Tethers may include any C 1 -C 100 carbon-containing moiety, (e.g. C 1 -C 75 , C 1 -C 50 , C 1 -C 20 , C 1 -C 10 ; C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , or C 10 ), and may have at least one nitrogen atom.
  • the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) group on the linker/tether, which may serve as a connection point for the ligand.
  • linkers/tethers underlined
  • linkers/tethers include TAP-(CH 2 ) n NH-; TAP- C(O)(CH 2 ) n NH-; TAP-NR'''(CH 2 ) n NH-, TAP-C(O)-(CH 2 ) n -C(O)-; TAP-C(O)-(CH 2 ) n - C(O)O-; TAP-C(O)-O-; TAP-C(O)-(CH 2 ) n -NH-C(O)-; TAP-C(O)-(CH 2 ) n -; TAP-C(O)-NH-; TAP-C(O)-; TAP-C(O)-; TAP-(CH 2 )
  • n is 5, 6, or 11.
  • the nitrogen may form part of a terminal oxyamino group, e.g., -ONH 2 , or hydrazino group, -NHNH 2 .
  • the linker/tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S.
  • Preferred tethered ligands may include, e.g., TAP-(CH 2 ) n NH(LIGAND); TAP- C(O)(CH 2 ) n NH(LIGAND); TAP -NR' ' ('C'H 2 ) n NH(LIGAND); TAP-(CH 2 ) n ONH(LIGAND); TAP-C(O)(CH 2 ) n ONH(LIGAND); TAP -NR' ' ('C'H 2 ) n ONH(LIGAND); TAP- (CH 2 ) n NHNH 2 (LIGAND ⁇ TAP-C(O)(CH 2 ) n NHNH 2 (LIGAND); TAP-
  • amino terminated linkers/tethers e.g., NH 2 , ONH 2 , NH 2 NH 2
  • amino terminated linkers/tethers e.g., NH 2 , ONH 2 , NH 2 NH 2
  • the tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S.
  • the double bond can be cis or trans or E or Z.
  • the linker/tether may include an electrophilic moiety, preferably at the terminal position of the linker/tether.
  • Exemplary electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g.
  • Preferred linkers/tethers include TAP-(CH 2 ) n CHO; TAP-C(O)(CH 2 ) n CHO; or TAP- NR'''(CH 2 ) n CHO, in which n is 1-6 and R''” is C 1 -C 6 alkyl; or TAP-(CH 2 ) n C(O)ONHS; TAP-C(O)(CH 2 ) nC(O)ONHS; or TAP-NR''''(CH 2 ) n C(O)ONHS, in which n is 1-6 and R”” is C 1 -C 6 alkyl; TAP-(CH 2 ) n C(O)OC 6 F 5 ; TAP-C(O)(CH 2 ) n C(O) OC 6 F 5 ; or TAP-NR'''(CH 2 ) nC(O)
  • the monomer can include a phthalimido group (K) at the terminal position of the linker/tether
  • other protected amino groups can be at the terminal position of the linker/tether, e.g., alloc, monomethoxy trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can be ortho-nitrophenyl or ortho, para- dinitrophenyl).
  • MMT monomethoxy trityl
  • Fmoc Fmoc
  • aryl sulfonyl e.g., the aryl portion can be ortho-nitrophenyl or ortho, para- dinitrophenyl.
  • At least one of the linkers/tethers can be a redox cleavable linker, an acid cleavable linker, an esterase cleavable linker, a phosphatase cleavable linker, a peptidase cleavable linker, or endosomal cleavable linker.
  • At least one of the linkers/tethers can be a reductively cleavable linker (e.g., a disulfide group).
  • At least one of the linkers/tethers can be an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group).
  • an acid cleavable linker e.g., a hydrazone group, an ester group, an acetal group, or a ketal group.
  • At least one of the linkers/tethers can be an esterase cleavable linker (e.g., an ester group).
  • at least one of the linkers/tethers can be a phosphatase cleavable linker (e.g., a phosphate group).
  • At least one of the linkers/tethers can be a peptidase cleavable linker (e.g., a peptide bond).
  • At least one of the linkers/tethers can be an endosomal cleavable linker (or a protease cleavable linker, e.g., a carbohydrate linker).
  • a carbohydrate linker is cleaved at least 1.25 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 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 tethers will have a linkage group that is cleaved at a preferred pH, thereby releasing the iRNA agent from a ligand (e.g., a targeting or cell- permeable ligand, such as cholesterol) inside the cell, or into the desired compartment of the cell.
  • a ligand e.g., a targeting or cell- permeable ligand, such as cholesterol
  • a chemical junction that links a ligand to an iRNA agent can include a disulfide bond.
  • a disulfide bond When the iRNA agent/ligand complex is taken up into the cell by endocytosis, the acidic environment of the endosome will cause the disulfide bond to be cleaved, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. 19: 1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002).
  • the ligand can be a targeting ligand or a second therapeutic agent that may complement the therapeutic effects of the iRNA agent.
  • a tether can include a linking group that is cleavable by a particular enzyme.
  • the type of linking group incorporated into a tether can depend on the cell to be targeted by the iRNA agent.
  • an iRNA agent that targets an mRNA in liver cells can be conjugated to a tether that includes an ester group. Liver cells are rich in esterases, and therefore the tether will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Cleavage of the tether releases the iRNA agent from a ligand that is attached to the distal end of the tether, thereby potentially enhancing silencing activity of the iRNA agent.
  • Tethers that contain peptide bonds can be conjugated to iRNA agents target to cell types rich in peptidases, such as liver cells and synoviocytes.
  • iRNA agent targeted to synoviocytes such as for the treatment of an inflammatory disease (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond.
  • 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, e.g., tissue the iRNA agent would be exposed to when administered to a subject.
  • tissue e.g., tissue the iRNA agent would be exposed to when administered to a subject.
  • 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.
  • 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).
  • the cleavable linker may be cleavable in various tissue and cell structures, e.g., in a homogenate, tritosome, cytosol, or endosome of any types of cells, such as in liver homogenates, liver tritosomes, liver lysosomes, liver cytosol, liver endosomes, brain homogenates, brain tritosomes, brain lysosomes, brain cytosol, or brain endosomes.
  • Redox Cleavable Linking Groups e.g., in a homogenate, tritosome, cytosol, or endosome of any types of cells, such as in liver homogenates, liver tritosomes, liver lysosomes, liver cytosol, liver endosomes, brain homogenates, brain tritosomes, brain lysosomes, brain cytosol, or brain endosomes.
  • One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation.
  • An example of reductively cleavable linking group is a disulphide 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 linking groups 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(S)(ORk)-O— , — O— P(S)(ORk)-O— , — O— P(S)(ORk)-
  • 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(S)(H)— O— , — O— P(S)(H)— O— , — S— P(O)(H)— O— , — S— P(O)(H)— O— , — S— P(O)(H)— O— , — S— P(O)(H)— O— , — S— P(O)(H)— O— , — S— P(
  • Acid cleavable linking groups 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, ketals, acetals, 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 linking groups 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 linking groups 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 cleavable linking groups have the general formula — NHCHR 1 C(O)NHCHR 2 C(O) — , where R 1 and R 2 are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
  • the linkers can also include biocleavable linkers that are nucleotide and non- nucleotide linkers, or combinations thereof, that connect two parts of a molecule.
  • a biocleavable linker may be used as part of the linking group L to connect the two oligonucleotides of the single-stranded oligonucleotide.
  • mere electrostatic or stacking interaction between two individual nucleotide sequences can represent a linker.
  • the non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and derivatives thereof, aliphatic, alicyclic, heterocyclic, and combinations thereof.
  • At least one of the linkers is a bio-cleveable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.
  • the cleavable linker (or the bio-cleavable linker) contains one or more carbohydrate (saccharide) moieties and/or a peptide linker.
  • the cleavable linker (or the bio-cleavable linker) may be used to connect two nucleotide sequences or oligonucleotides, connect a nucleotide sequence or an oligonucleotide with a ligand, or connect a ligand and endosomal cleavable agent.
  • the bio-cleavable carbohydrate linker has one or more of the following features: i) the bio-cleavable carbohydrate linker may have 1 to 10 saccharide units, ii) the saccharide moieties have at least one anomeric linkage capable of connecting two nucleotide sequences or oligonucleotides, iii) when two or more saccharides are present, these nucleotide sequences or oligonucleotides can be linked via 1-3, 1-4, or 1-6 sugar linkages, iv) when two or more saccharides are present, these nucleotide sequences or oligonucleotides may also be linked via alkyl chains.
  • bio-cleavable linkers include:
  • the cleavable linker (or the bio-cleavable linker) is an endosomal cleavable linker comprising one or more saccharide units independently selected from the following groups:
  • the endosomal cleavable linker comprises two or more of the above saccharide units.
  • the endosomal cleavable linker comprises 1-10 of the saccharide units.
  • the endosomal cleavable linker comprises 2-10 of the saccharide units.
  • the saccharide units in the endosomal cleavable linker are selected from the group consisting of Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316 and Q317.
  • the endosomal cleavable linker comprises 2, 3, or 4 of the saccharide units.
  • the saccharide units are selected from the group consisting of Q303, Q304, Q305, Q306, Q312, Q313, Q314, Q315, Q316 and Q317.
  • the saccharide units may be Q304.
  • the endosomal cleavable linker comprises
  • the endosomal cleavable linker further comprises
  • the endosomal cleavable linker comprises: -Q198Q48Q303Q303Q48-, -Q198Q303Q48Q303-, -Q198Q48Q303Q303Q48-, -Q198Q303Q48Q303-, -Q198Q303Q303Q303-, -Q198Q303Q303-, -Q198Q303Q303-, -Q198Q304Q304Q304-, -Q198Q304Q304Q304-, -Q198Q304Q304-, -Q198Q304Q304-, -Q198Q304Q304-, -Q198Q48Q303Q48-, -Q198Q303Q48Q303-, -Q198Q303Q303-, -Q48Q303Q303Q48-, or -Q303Q48Q303-.
  • the linking group L connecting the two oligonucleotides of the single-stranded oligonucleotide contains one or more carriers.
  • one or more ligands are conjugated to the single-stranded oligonucleotide via one or more carriers.
  • the carrier may replace one or more nucleotide(s).
  • the carrier can be a cyclic group or an acyclic group.
  • the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] di oxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalinyl.
  • the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.
  • the carrier replaces one or more nucleotide(s) in the internal position(s) of a nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 1 and/or Z 2 ).
  • a ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS).
  • the carrier can be a cyclic or acyclic moiety and include two “backbone attachment points'' (e.g., hydroxyl groups) and a ligand.
  • the ligand can be directly attached to the carrier or indirectly attached to the carrier by an intervening linker/tether, as described above.
  • the ligand-conjugated monomer subunit may be the 5' or 3' terminal subunit of a nucleotide sequence of the single-stranded oligonucleotide (e.g., Z 1 and/or Z 2 ), i.e., one of the two “W'' groups may be a hydroxyl group, and the other “W'' group may be a chain of two or more unmodified or modified ribonucleotides.
  • the ligand-conjugated monomer subunit may occupy an internal position, and both “W'' groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be present in a single-stranded oligonucleotide.
  • Cyclic sugar replacement-based monomers e.g., sugar replacement-based ligand- conjugated monomers
  • the carriers may have the general formula (LCM-2) provided below (In that structure preferred backbone attachment points can be chosen from R 1 or R 2 ; R 3 or R 4 ; or R 9 and R 10 if Y is CR 9 R 10 (two positions are chosen to give two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 )).
  • Preferred tethering attachment points include R 7 ; R 5 or R 6 when X is CH 2 .
  • the carriers are described below as an entity, which can be incorporated into a strand.
  • the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R 1 or R 2 ; R 3 or R 4 ; or R 9 or R 10 (when Y is CR 9 R 10 ), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone.
  • one of the above-named R groups can be - CH 2 -, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.
  • X is N(CO)R 7 , NR 7 or CH 2 ;
  • Y is NR 8 , O, S, CR 9 R 10 ;
  • Z is CR 11 R 12 or absent
  • Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , or (CH 2 ) n OR b , provided that at least two of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 are OR a and/or (CH 2 ) n OR b ;
  • R 5 , R 6 , R 11 , and R 12 is, independently, a ligand, H, C 1 -C 6 alkyl optionally substituted with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 ;
  • R 7 can be a ligand, e.g., R 7 can be R d , or R 7 can be a ligand tethered indirectly to the carrier, e.g., through a tethering moiety, e.g., C 1 -C 20 alkyl substituted with NR c R d ; or C 1 -C 20 alkyl substituted with NHC(O)R d ;
  • R 8 is H or C 1 -C 6 alkyl
  • R 13 is hydroxy, C 1 -C4 alkoxy, or halo
  • R 14 is NR c R 7 ;
  • R 15 is C 1 -C 6 alkyl optionally substituted with cyano, or C 2 -C 6 alkenyl
  • R 16 is C 1 -C 10 alkyl
  • R 17 is a liquid or solid phase support reagent
  • L is -C(O)(CH 2 ) q C(O)-, or -C(O)(CH 2 ) q S-;
  • R a is a protecting group, e.g., CAr 3 ; (e.g., a dimethoxytrityl group) or Si(X 5' )(X 5” )(X 5" ' ) in which (X 5' ),(X 5” ), and (X 5" ' ) are as described elsewhere.
  • R b is P(O)(O )H, P(OR 15 )N(R 16 ) 2 or L-R 17 ;
  • R c is H or C 1 -C 6 alkyl
  • R d is H or a ligand
  • Each Ar is, independently, C 6 -C 10 aryl optionally substituted with C 1 -C 4 alkoxy; n is 1-4; and q is 0-4.
  • the carrier may be based on the pyrroline ring system or the 4-hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent
  • OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the five- membered ring (-CH 2 OFG 1 in D).
  • OFG 2 is preferably attached directly to one of the carbons in the five-membered ring (-OFG 2 in D).
  • -CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or -CH 2 OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4.
  • CH 2 OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • -CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-4.
  • the pyrroline- and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • CH 2 OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH 2 OFG 1 and OFG 2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa).
  • the tethering attachment point is preferably nitrogen.
  • Preferred examples of carrier D include the following:
  • the carrier may be based on the piperidine ring system
  • -(CH 2 ) n OFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4.
  • -(CH 2 ) n OFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., - (CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; -(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-3.
  • the piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • the tethering attachment point is preferably nitrogen.
  • the carrier may be based on the piperazine ring system
  • OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (-CH 2 OFG 1 in F or G).
  • OFG 2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG 2 in F or G).
  • -CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or vice versa.
  • CH 2 OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • the piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • CH 2 OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • the tethering attachment point is preferably nitrogen in both F and G.
  • -(CH 2 ) n OFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5.
  • -(CH 2 ) n OFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; - (CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; -(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-3; -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-5; or -(CH 2 ) n OFG 1 may be attached to C-5 and OFG 2 may be attached to C-4.
  • the decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • the centers bearing CH 2 OFG 1 and OFG 2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa).
  • the substituents at C-l and C-6 are trans with respect to one another.
  • the tethering attachment point is preferably C-6 or C-7.
  • Other carriers may include those based on 3 -hydroxyproline (J). .
  • -(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH 2 OFG 1 and OFG 2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa).
  • the tethering attachment point is preferably nitrogen.
  • Acyclic sugar replacement-based monomers e.g., sugar replacement-based ligand-conjugated monomers
  • RRMS ribose replacement monomer subunit
  • Preferred acyclic carriers can have formula LCM-3 or LCM-4:
  • each of x, y, and z can be, independently of one another, 0, 1, 2, or 3.
  • the tertiary carbon can have either the R or S configuration.
  • x is zero and y and z are each 1 in formula LCM-3 (e.g., based on serinol), and y and z are each 1 in formula LCM-3.
  • Each of formula LCM-3 or LCM-4 below can optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl.
  • the single-stranded oligonucleotide comprises one or more ligands conjugated to the 5' end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the ligand is conjugated to the 5'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ) via a carrier and/or linker. In one embodiment, the ligand is conjugated to the 5'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ) via a carrier of a formula: R is a ligand.
  • the single-stranded oligonucleotide comprises one or more ligands conjugated to the 3' end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the ligand is conjugated to the 3'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ) via a carrier and/or linker. In one embodiment, the ligand is conjugated to the 3'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ) via a carrier of a formula
  • R is a ligand
  • the ligand is conjugated to a nucleotide sequence (e.g., Z 1 and/or Z 2 ) via one or more linkers (tethers) and/or a carrier. In one embodiment, the ligand is conjugated to a nucleotide sequence (e.g., Z 1 and/or Z 2 ) via one or more linkers (tethers).
  • the ligand is conjugated to the 5' end or 3' end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ) via a cyclic carrier, optionally via one or more intervening linkers (tethers).
  • a nucleotide sequence e.g., Z 1 and/or Z 2
  • tethers optionally via one or more intervening linkers
  • the ligand is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • Internal positions of a nucleotide sequence refer to the nucleotide on any position of the nucleotide sequence, except the terminal position from the 3' end and 5' end of the nucleotide sequence (e.g., excluding 2 positions: position 1 counting from the 3' end and position 1 counting from the 5' end).
  • the ligand is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ), which include all positions except the terminal two positions from each end of the nucleotide sequence (e.g., excluding 4 positions: positions 1 and 2 counting from the 3' end and positions 1 and 2 counting from the 5' end).
  • nucleotide sequence e.g., Z 1 and/or Z 2
  • the lipophilic moiety is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ), which include all positions except the terminal three positions from each end of the nucleotide sequence (e.g., excluding 6 positions: positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).
  • nucleotide sequence e.g., Z 1 and/or Z 2
  • Z 1 and/or Z 2 include all positions except the terminal three positions from each end of the nucleotide sequence (e.g., excluding 6 positions: positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).
  • the ligand is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ), except the cleavage site region of a nucleotide sequence, for instance, the ligand is not conjugated to positions 9-12 counting from the 5'-end of a nucleotide sequence, for example, the ligand is not conjugated to positions 9-11 counting from the 5'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the internal positions exclude positions 11-13 counting from the 3 -'end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the internal positions exclude positions 12-14 counting from the 5 '-end of a nucleotide sequence.
  • the ligand is conjugated to one or more internal positions on at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ), which exclude positions 11-13 on a nucleotide sequence, counting from the 3 '-end, and positions 12-14 on a nucleotide sequence (e.g., Z 1 and/or Z 2 ), counting from the 5'-end.
  • nucleotide sequence e.g., Z 1 and/or Z 2
  • one or more ligands are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on a nucleotide sequence (e.g., Z 1 and/or Z 2 ), and positions 6-10 and 15-18 on a nucleotide sequence (e.g., Z 1 and/or Z 2 ), counting from the 5' end.
  • one or more ligands are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on a nucleotide sequence (e.g., Z 1 and/or Z 2 ), and positions 15 and 17 on a nucleotide sequence (e.g., Z 1 and/or Z 2 ), counting from the 5' end.
  • a nucleotide sequence e.g., Z 1 and/or Z 2
  • positions 15 and 17 on a nucleotide sequence e.g., Z 1 and/or Z 2
  • the ligand is conjugated to a nucleobase, sugar moiety, or intemucleosidic linkage of the single-stranded oligonucleotide.
  • the single-stranded oligonucleotide is further modified by covalent attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached single-stranded oligonucleotide including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound.
  • conjugate groups 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.
  • the single-stranded oligonucleotide further comprises a targeting ligand that targets a receptor which mediates delivery to a specific CNS tissue.
  • a targeting ligand that targets a receptor which mediates delivery to a specific CNS tissue.
  • These targeting ligands can be conjugated in combination with the lipophilic moiety to enable specific local (e.g., intrathecal) and systemic delivery.
  • Exemplary targeting ligands that targets the receptor mediated delivery to a CNS tissue are peptide ligands such as Angiopep-2, lipoprotein receptor related protein (LRP) ligand, bEnd.3 cell binding ligand; transferrin receptor (TfR) ligand (which can utilize iron transport system in brain and cargo transport into the brain parenchyma); manose receptor ligand (which targets olfactory ensheathing cells, glial cells), glucose transporter protein, and LDL receptor ligand.
  • LRP lipoprotein receptor related protein
  • TfR transferrin receptor
  • manose receptor ligand which targets olfactory ensheathing cells, glial cells
  • glucose transporter protein and LDL receptor ligand.
  • the single-stranded oligonucleotide further comprises a targeting ligand that targets a receptor which mediates delivery to a specific ocular tissue.
  • a targeting ligand that targets a receptor which mediates delivery to a specific ocular tissue.
  • These targeting ligands can be conjugated in combination with the lipophilic moiety to enable specific local (e.g., intravitreal) and systemic delivery.
  • lipophilic ligands such as all-trans retinol (which targets the retinoic acid receptor ); RGD peptide (which targets retinal pigment epithelial cells), such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH (SEQ ID NO: 1) or
  • Preferred conjugate groups amenable to the present invention include lipid moi eties 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 moi eties 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
  • a phospholipid e.g., di-hexadecyl-rac-glycerol or triethylammonium-l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl.
  • 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-hydroxypropyl)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
  • 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, geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, 03- (oleoyl)cholenic acid, dimethoxy
  • 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- ⁇ B, taxon, vincristine, vinblastine, cytochalasin, nocodazole
  • Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; ⁇ , ⁇ , 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, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H 2 A 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 branched polyamines, 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,
  • 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, 1,2- dileoyl-sn-3 -phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z, 12Z)-octadeca-9, 12-dienyl)-1,3-dioxolan-4- yl)methanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-
  • Exemplary cell permeation peptides include, but are not limited to, [0419]
  • NH 2 alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid
  • NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE NH 2 ; 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. GalNAc 2 and Gal N Ac 3 (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); D-mannose, multivalent mannose, multivalent lactose, N-acetyl- glucosamine, Glucose, multivalent Glucose, 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 the composition of the invention.
  • 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).
  • 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 intemucleotide linkages in PK modulating oligonucleotide are phosphorothioate and/or phosphorodithioates linkages.
  • aptamers that bind serum components 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, such 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 or tethered ligand can be present on a monomer when said monomer is incorporated into a component of the single-stranded oligonucleotide.
  • the ligand can be incorporated via coupling to a “precursor'' monomer after said “precursor'' monomer has been incorporated into a component of the single-stranded oligonucleotide.
  • a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., monomer-linker-NH 2 can be incorporated into a component of the single- stranded oligonucleotide.
  • a ligand having an electrophilic group e.g., a pentafluorophenyl ester or aldehyde group
  • a ligand having an electrophilic group can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer's tether.
  • a monomer having a chemical group suitable for taking part in Click Chemistry reaction can be incorporated e.g., an azide or alkyne terminated tether/linker.
  • a ligand having complementary chemical group e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
  • ligand can be conjugated to nucleobases, sugar moieties, or intemucleosidic linkages of the single-stranded oligonucleotide.
  • Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms.
  • the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety.
  • Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position.
  • the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety.
  • Conjugation to sugar moieties of nucleosides can occur at any carbon atom.
  • Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as in an abasic residue. Intemucleosidic linkages can also bear conjugate moieties.
  • the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom.
  • the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
  • an oligonucleotide is attached to a conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, and the like) on the oligonucleotide with a reactive group on the conjugate moiety.
  • a reactive group e.g., OH, SH, amine, carboxyl, aldehyde, and the like
  • one reactive group is electrophilic and the other is nucleophilic.
  • an electrophilic group can be a carbonyl-containing functionality and a nucleophilic group can be an amine or thiol.
  • Methods for conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in the literature such as, for example, in Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.
  • the single-stranded oligonucleotide further comprises one or more targeting ligands that target a liver tissue.
  • at least one of the targeting ligands is a carbohydrate-based ligand.
  • the carbohydrate- based ligand is an ASGPR ligand.
  • at least one of the targeting ligands is a GalNAc-based conjugate.
  • the carbohydrate-based ligand is any one of the ligands listed in Table 2, Table 2A, Table 3, Table 3A, Table 4, or Table 4A of WO2015/006740, which is incorporated herein by reference in its entirety.
  • the linkers including branched linkers such as a bivalent or trivalent branched linker for attaching these carbohydrate-based ligands include the linker(s) listed in Table 1 or Table 1A and the spacer(s) listed in Table 5 of WO2015/006740, which is incorporated herein by reference in its entirety.
  • the GalNAc-based conjugate is a GalNAc analog containging a S or N atom, or a -CH 2 - group in the glycosidic linkage to change a metagolically labile glycosidic linkage to a metabolically stable glycosidic linkage, e.g., having “O'' in the glycosidic linkage being replaced by S or N atom, or a -CH 2 - group, as shown in the scheme below.
  • the GalNAc-based conjugate is a GalNAc analog having one of the following structures: ' '
  • GalNAc analogs listed in the above table may be prepared using the methods described in WO2015/006740, which is incorporated herein by reference in its entirety.
  • the single-stranded oligonucleotide further comprises a ligand having a structure shown below: wherein: L G is independently for each occurrence a ligand, e.g., carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, polysaccharide; and
  • Z', Z'', Z''' and Z'''' are each independently for each occurrence O or S.
  • the single-stranded oligonucleotide comprises a ligand of
  • Q and Q' are independently for each occurrence is absent, -(P 7 -Q 7 -R 7 ) p -T 7 - or -T 7 - Q 7 -T 7 -B-T 8 -Q 8 -T 8 ;
  • P 2A P 2B P 3A P 3B P 4A P 4B P 5A P 5B P 5C P 7 ,T 2A ,T 2B ,T 3 A ,T 3B , T 4A ,T 4B , T 4A , T 5B , T 5C , T 7 , T 7' , T 8 and T 8' are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O;
  • B is -CH 2 -N(B L )-CH 2 -;
  • B L is -T B -Q B -T B '-R X;
  • T B and T B are each independently for each occurrence absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH 2 , CH 2 NH or CH 2 O;
  • R x is a lipophile (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasacchari
  • R 1 , R 2 , R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C , R 7 are each independently for each occurrence absent, NH, O, S, CH 2 , C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-,
  • L 1 , L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C are each independently for each occurrence a carbohydrate, e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide;
  • R' and R'' are each independently H, C 1 -C 6 alkyl, OH, SH, or N(R N ) 2 ;
  • R N is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl or benzyl;
  • R a is H or amino acid side chain
  • Z', Z'', Z''' and Z'''' are each independently for each occurrence O or S; p represents independently for each occurrence 0-20.
  • the ligand can be conjugated to the single-stranded oligonucleotide via a linker or carrier, and because the linker or carrier can contain a branched linker, the single-stranded oligonucleotide can then contain multiple ligands via the same or different backbone attachment points to the carrier, or via the branched linker(s).
  • the branchpoint of the branched linker may be a bivalent, trivalent, tetravalent, pentavalent, or hexavalent atom, or a group presenting such multiple valencies.
  • the branchpoint is -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(O)N(Q)-C, - OC(O)N(Q)-C, -N(Q)C(O)-C, or -N(Q)C(O)O-C; wherein Q is independently for each occurrence H or optionally substituted alkyl.
  • the branchpoint is glycerol or glycerol derivative.
  • the ASGPR ligand conjugated to the single-stranded oligonucleotide is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a ligand of: Exemplary ligand monomers
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of: [0460] In certain embodiments, the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide of the invention comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • both L 2A and L 2B are the same. In some embodiments, both L 2A and L 2B are different.
  • both L 3A and L 3B are the same. In some embodiments, both L 3A and L 3B are different.
  • both L 4A and L 4B are the same. In some embodiments, both L 4A and L 4B are different.
  • all of L 5A , L 5B and L 5C are the same. In some embodiments, two of L 5A , L 5B and L 5C are the same. In some embodiments, L 5A and L 5B are the same. In some embodiments, L 5A and L 5C are the same. In some embodiments, L 5B and L 5C are the same.
  • the single-stranded oligonucleotide comprises a monomer
  • the single-stranded oligonucleotide comprises a monomer of: [0483] In certain embodiments, the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of:
  • Y is O or S, and n is 1-6.
  • the single-stranded oligonucleotide comprises a monomer , wherein Y is O or S, n is 1-6, R is hydrogen or nucleic acid, and R' is nucleic acid.
  • the single-stranded oligonucleotide comprises a monomer of , wherein Y is O or S, and n is 1-6. [0487] In certain embodiments, the single-stranded oligonucleotide comprises a monomer of structure: , wherein Y is O or S, n is 2-6, x is
  • A is H or a phosphate linkage.
  • the single-stranded oligonucleotide comprises at least 1, 2, '
  • the single-stranded oligonucleotide comprises a monomer ' ' of: , wherein X is O or S.
  • the single-stranded oligonucleotide comprises a monomer of: , wherein x is 1-12. [0491] In some embodiments, the single-stranded oligonucleotide comprises a monomer of: wherein R is OH or NHCOCH 3 .
  • the single-stranded oligonucleotide comprises a monomer of: wherein R is OH or NHCOCH 3 .
  • the single-stranded oligonucleotide comprises a monomer of: wherein R is O or S.
  • the single-stranded oligonucleotide comprises a monomer of: wherein R is OH or NHCOCH 3 . [0495] In certain embodiments, the single-stranded oligonucleotide comprises a monomer of:
  • the single-stranded oligonucleotide comprises a monomer of: , wherein R is OH or NHCOCH 3 .
  • the single-stranded oligonucleotide comprises a monomer of: , wherein R is OH or NHCOCH 3 .
  • the single-stranded oligonucleotide comprises a monomer ' of: , wherein R is OH or NHCOCH 3 .
  • the single-stranded oligonucleotide comprises a monomer of: , wherein R is OH or NHCOCH 3 .
  • the single-stranded oligonucleotide comprises a monomer of:
  • X and Y are each independently for each occurrence H, a protecting group, a phosphate group, a phosphodiester group, an activated phosphate group, an activated phosphite group, a phosphoramidite, a solid support, - P(Z’)(Z'')O-nucleoside, -P(Z’)(Z'')O-oligonucleotide, a lipid, a PEG, a steroid, a polymer, a nucleotide, a nucleoside, or an oligonucleotide; and Z' and Z'' are each independently for each occurrence O or S.
  • the single-stranded oligonucleotide is conjugated with a ligand of:
  • the single-stranded oligonucleotide comprises a ligand of: '
  • the single-stranded oligonucleotide comprises a monomer of: or
  • At least one of the ligands conjugated to the single- stranded oligonucleotide is a lipophilic moiety.
  • lipophile'' or lipophilic moiety'' broadly refers to any compound or chemical moiety having an affinity for lipids.
  • One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, logK ow , where K ow is the ratio of a chemical's concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium.
  • the octanol-water partition coefficient is a laboratory- measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first- principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci.
  • a chemical substance is lipophilic in character when its logK ow exceeds 0.
  • the lipophilic moiety possesses a logK ow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10.
  • the logK ow of 6-amino hexanol for instance, is predicted to be approximately 0.7.
  • the logK ow of cholesteryl N- (hexan-6-ol) carbamate is predicted to be 10.7.
  • the lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logK ow ) value of the lipophilic moiety.
  • the hydrophobicity of the single-stranded oligonucleotide, conjugated to one or more lipophilic moieties can be measured by its protein binding characteristics.
  • the unbound fraction in the plasma protein binding assay of the single- stranded oligonucleotide can be determined to positively correlate to the relative hydrophobicity of the single-stranded oligonucleotide, which can positively correlate to the silencing activity of the single-stranded oligonucleotide.
  • the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein.
  • ESA electrophoretic mobility shift assay
  • conjugating the lipophilic moieties to the internal position(s) of the single- stranded oligonucleotide provides optimal hydrophobicity for the enhanced in vivo delivery of single-stranded oligonucleotide.
  • the lipophilic moiety is an aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compound, such as a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon.
  • the lipophilic moiety may generally comprise a hydrocarbon chain, which may be cyclic or acyclic.
  • the hydrocarbon chain may comprise various substituents and/or one or more heteroatoms, such as an oxygen or nitrogen atom.
  • Such lipophilic aliphatic moieties include, without limitation, saturated or unsaturated C 4 -C 30 hydrocarbon (e.g., C 6 -C 18 hydrocarbon or C 14 -C 24 hydrocarbon), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 terpenes, C15 sesquiterpenes, C20 diterpenes, C30 triterpenes, and C40 tetraterpenes), and other polyalicyclic hydrocarbons.
  • C 4 -C 30 hydrocarbon e.g., C 6 -C 18 hydrocarbon or C 14 -C 24 hydrocarbon
  • saturated or unsaturated fatty acids e.g., waxe.g., monohydric alcohol esters of fatty acids and fatty diamides
  • terpenes e.g., C 10 terpenes, C15 sesquiterpenes
  • the lipophilic moiety may contain a C4- C 30 hydrocarbon chain (e.g., C 4 -C 30 alkyl or alkenyl).
  • the lipophilic moiety contains a saturated or unsaturated C 6 -C 18 hydrocarbon chain (e.g., a linear C 6 -C 18 alkyl or alkenyl) or a saturated or unsaturated C 14 -C 24 hydrocarbon (e.g., a linear C 14 -C 24 alkyl or alkenyl).
  • the lipophilic moiety contains a saturated or unsaturated C 16 hydrocarbon chain (e.g., a linear C 16 alkyl or alkenyl) or a saturated or unsaturated C 22 hydrocarbon chain (e.g., a linear C 22 alkyl or alkenyl).
  • a saturated or unsaturated C 16 hydrocarbon chain e.g., a linear C 16 alkyl or alkenyl
  • a saturated or unsaturated C 22 hydrocarbon chain e.g., a linear C 22 alkyl or alkenyl
  • the lipophilic moiety may be attached to the single-stranded oligonucleotide by any method known in the art, including via a functional grouping already present in the lipophilic moiety or introduced into the single-stranded oligonucleotide, such as a hydroxy group (e.g., — CO — CH 2 — OH).
  • a functional grouping already present in the lipophilic moiety or introduced into the single-stranded oligonucleotide such as a hydroxy group (e.g., — CO — CH 2 — OH).
  • the functional groups already present in the lipophilic moiety or introduced into the single-stranded oligonucleotide include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.
  • Conjugation of the single-stranded oligonucleotide and the lipophilic moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy and an alkyl group R — , an alkanoyl group RCO — or a substituted carbamoyl group RNHCO — .
  • the alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated).
  • Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like.
  • the lipophilic moiety is conjugated to the single-stranded oligonucleotide via a linker a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.
  • a linker a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.
  • the lipophilic moiety is a steroid, such as sterol.
  • Steroids are polycyclic compounds containing a perhydro-l,2-cyclopentanophenanthrene ring system.
  • Steroids include, without limitation, bile acids (e.g., cholic acid, deoxycholic acid and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone.
  • a “cholesterol derivative'' refers to a compound derived from cholesterol, for example by substitution, addition or removal of substituents.
  • the lipophilic moiety is an aromatic moiety.
  • aromatic'' refers broadly to mono- and polyaromatic hydrocarbons.
  • Aromatic groups include, without limitation, C 6 -C 14 aryl moieties comprising one to three aromatic rings, which may be optionally substituted; “aralkyl'' or “arylalkyl'' groups comprising an aryl group covalently linked to an alkyl group, either of which may independently be optionally substituted or unsubstituted; and “heteroaryl'' groups.
  • heteroaryl'' refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14 ⁇ electrons shared in a cyclic array, and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).
  • a “substituted'' alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having between one and about four, preferably between one and about three, more preferably one or two, non-hydrogen substituents.
  • Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.
  • the lipophilic moiety is an aralkyl group, e.g., a 2- arylpropanoyl moiety.
  • the structural features of the aralkyl group are selected so that the lipophilic moiety will bind to at least one protein in vivo.
  • the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins.
  • the structural features of the aralkyl group promote binding to albumin, an immunoglobulin, a lipoprotein, a-2- macroglubulin, or a- 1-glycoprotein.
  • the ligand is naproxen or a structural derivative of naproxen.
  • Procedures for the synthesis of naproxen can be found in U.S. Pat. No. 3,904,682 and U.S. Pat. No. 4,009,197, which are herein incorporated by reference in their entirety.
  • Naproxen has the chemical name (S)-6-Methoxy-a-methyl-2-naphthaleneacetic acid and the structure is is
  • the ligand is ibuprofen or a structural derivative of ibuprofen.
  • Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No. 3,228,831, which are herein incorporated by reference in their entirety.
  • the structure of ibuprofen is
  • suitable lipophilic moieties include lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,
  • the lipophilic moiety is a C 6 -C 30 acid (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodcanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7, 10, 13, 16, 19- docosahexanoic acid, vitamin A, vitamin E, cholesterol etc.) or a C 6 -C 30 alcohol (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodcanol, tridecanol,
  • more than one lipophilic moieties can be incorporated into the single-stranded oligonucleotide, particularly when the lipophilic moiety has a low lipophilicity or hydrophobicity.
  • two or more lipophilic moieties are incorporated into the same strand of the single-stranded oligonucleotide.
  • each strand of the single-stranded oligonucleotide has one or more lipophilic moieties incorporated.
  • two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, same sugar moiety, or same intemucleosidic linkage) of the single-stranded oligonucleotide.
  • This can be achieved by, e.g., conjugating the two or more lipophilic moieties via a carrier, and/or conjugating the two or more lipophilic moieties via a branched linker, and/or conjugating the two or more lipophilic moieties via one or more linkers, with one or more linkers linking the lipophilic moieties consecutively.
  • the lipophilic moiety may be conjugated to the single-stranded oligonucleotide via a direct attachment to the ribosugar of the single-stranded oligonucleotide.
  • the lipophilic moiety may be conjugated to the single-stranded oligonucleotide via a linker or a carrier.
  • the lipophilic moiety may be conjugated to the single- stranded oligonucleotide via one or more linkers (tethers).
  • the lipophilic moiety is conjugated to the single-stranded oligonucleotide via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.
  • a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.
  • target nucleic acid'' refers to any nucleic acid molecule the expression or activity of which is capable of being modulated by an siRNA compound.
  • Target nucleic acids include, but are not limited to, RNA (including, but not limited to pre- mRNA and mRNA or portions thereof) transcribed from DNA encoding a target protein, and also cDNA derived from such RNA, and miRNA.
  • the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state.
  • a target nucleic acid can be a nucleic acid molecule from an infectious agent.
  • iRNA'' refers to an agent that mediates the targeted cleavage of an RNA transcript. These agents associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). Agents that are effective in inducing RNA interference are also referred to as siRNA, RNAi agent, or iRNA agent, herein. Thus, these terms can be used interchangeably herein.
  • RISC RNAi-induced silencing complex
  • siRNA RNAi agent
  • iRNA agent cytoplasmic multi-protein complex
  • iRNA agent agents that are effective in inducing RNA interference
  • the term iRNA includes microRNAs and pre-microRNAs.
  • the “compound'' or “compounds'' of the invention as used herein also refers to the iRNA agent, and can be used interchangeably with the iRNA agent.
  • the iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene.
  • nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an iRNA agent.
  • ribonucleotide'' or nucleotide'' can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.
  • the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA.
  • RNAi cleavage product thereof e.g., mRNA.
  • Complementarity, or degree of homology with the target strand is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more, or for example, 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA).
  • the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double stranded character of the molecule.
  • iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed siRNA agents or shorter iRNA agents herein.
  • siRNA agent or shorter iRNA agent'' refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60, 50, 40, or 30 nucleotide pairs.
  • the siRNA agent, or a cleavage product thereof can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, wherein the target may comprise an endogenous or pathogen target RNA.
  • a “single-stranded oligonucleotide'' or “single strand iRNA agent'' as used herein, is an oligonucleotide or iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin, dumbbell, or pan-handle structure. Single-stranded oligonucleotide or iRNA agent may be antisense with regard to the target molecule.
  • a single-stranded oligonucleotide or iRNA agent may be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA.
  • a single-stranded oligonucleotide or iRNA agent is at least 14, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.
  • the single-stranded oligonucleotide contains two oligonucleotides, connected by a linking group.
  • a loop refers to a region of an oligonucleotide or iRNA strand that is unpaired with the opposing nucleotide in the duplex when a section of the oligonucleotide or the iRNA strand forms base pairs with another strand or with another section of the same strand.
  • Hairpin oligonucleotides or iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs.
  • the duplex region will may be equal to or less than 200, 100, or 50, in length. In certain embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
  • the hairpin may have a single strand overhang or terminal unpaired region, in some embodiments at the 3', and in certain embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 2-3 nucleotides in length.
  • RNAi activity refers to gene silencing by an oligonucleotide, nucleic acid agent, or iRNA agent.
  • gene silencing by an oligonucleotide, nucleic acid agent, or iRNA agent refers to a decrease in the mRNA level in a cell for a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% up to and including 100%, and any integer in between of the mRNA level found in the cell without the presence of the miRNA or RNA interference molecule.
  • the mRNA levels are decreased by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to and including 100% and any integer in between 5% and 100%.”
  • modulate gene expression'' means that expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate'' can mean “inhibit,'' but the use of the word “modulate'' is not limited to this definition.
  • gene expression modulation happens when the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more different from that observed in the absence of the siRNA.
  • the % and/or fold difference can be calculated relative to the control or the non- control, for example, or
  • inhibitor'' As used herein, the term “inhibit'', “down-regulate'', or “reduce'' in relation to gene expression, means that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of modulator.
  • the gene expression is down-regulated when expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced at least 10% lower relative to a corresponding non-modulated control, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or most preferably, 100% (i.e., no gene expression).
  • the term “increase'' or “up-regulate'' in relation to gene expression” means that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is increased above that observed in the absence of modulator.
  • the gene expression is up-regulated when expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is increased at least 10% relative to a corresponding non-modulated control, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more.
  • the term "increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • reduced or “reduce” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • a double-stranded nucleic acid agent comprises two oligonucleotide strands that are sufficiently complementary to hybridize to form a duplex structure.
  • the duplex structure is between 8 and 30, between 15 and 30, between 18 and 25, between 19 and 24, or between 19 and 21 base pairs in length.
  • longer double-stranded nucleic acid agent of between 25 and 30 base pairs in length are preferred.
  • shorter double-stranded nucleic acid agent of between 10 and 15 base pairs in length are preferred.
  • the double-stranded nucleic acid agent is at least 21 nucleotides long.
  • antisense strand'' or “antisense oligonucleotide'' as used herein refers to an oligomeric compound that is substantially or 100% complementary to a target sequence of interest.
  • antisense strand includes the antisense region of both oligomeric compounds that are formed from two separate strands, as well as unimolecular oligomeric compounds that are capable of forming hairpin or dumbbell type structures.
  • antisense strand'' and guide strand'' are used interchangeably herein.
  • sense strand'' refers to an oligomeric compound that has the same nucleoside sequence, in whole or in part, as a target sequence such as a messenger RNA or a sequence of DNA.
  • target sequence such as a messenger RNA or a sequence of DNA.
  • sense strand'' and passenger strand'' are used interchangeably herein.
  • 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.
  • 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 Syrnp. 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.
  • the double-stranded region of a double-stranded nucleic acid agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.
  • the first oligonucleotide of a double-stranded nucleic acid agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the second oligonucleotide of a double-stranded nucleic acid agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the first and second oligonucleotides of the double-stranded nucleic acid agent are each 15 to 30 nucleotides in length.
  • the first and second oligonucleotides of the double-stranded nucleic acid agent are each 19 to 25 nucleotides in length.
  • the first and second oligonucleotides of the double-stranded nucleic acid agent are each 21 to 23 nucleotides in length.
  • one oligonucleotide has at least one stretch of 1-5 single- stranded nucleotides in the double-stranded region.
  • stretch of single-stranded nucleotides in the double-stranded region'' is meant that there is present at least one nucleotide base pair at both ends of the single-stranded stretch.
  • both strands have at least one stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double stranded region.
  • both strands have a stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single- stranded nucleotides in the double stranded region
  • such single-stranded nucleotides can be opposite to each other (e.g., a stretch of mismatches) or they can be located such that the second oligonucleotide has no single-stranded nucleotides opposite to the single-stranded nucleotide of the first oligonucleotide and vice versa (e.g., a single-stranded loop).
  • the single-stranded nucleotides are present within 8 nucleotides from either end, for example 8, 7, 6, 5, 4, 3, or 2 nucleotides from either the 5' or 3' end of the region of complementarity between the two oligonucleotides.
  • the double-stranded nucleic acid agent comprises a single- stranded overhang on at least one of the termini.
  • the single-stranded overhang is 1, 2, or 3 nucleotides in length.
  • the second oligonucleotide of the double-stranded nucleic acid agent is 21- nucleotides in length
  • the first oligonucleotide is 23 -nucleotides in length
  • the first and second oligonucleotides form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single-stranded overhangs at the 3 -'end.
  • each oligonucleotide of the double-stranded nucleic acid agent has a ZXY structure, such as is described in PCT Publication No. 2004080406, which is hereby incorporated by reference in its entirety.
  • the two nucleotide sequences can be linked together to form a long strand.
  • the two nucleotide sequences can be linked together by an oligonucleotide linker including, but not limited to, (N) n ; wherein N is independently a modified or unmodified nucleotide and n is 3-23.
  • n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10.
  • the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide.
  • N is a modified or unmodified nucleotide
  • R is a modified or unmodified purine nucleotide.
  • Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker.
  • the two nucleotide sequences can also be linked together by a non-nucleotide based linker, e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker.
  • two strands specifically hybridize when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.
  • stringent hybridization conditions'' or “stringent conditions'' refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences.
  • Stringent conditions are sequence-dependent and will be different in different circumstances, and “stringent conditions'' under which antisense compounds hybridize to a target sequence are determined by the nature and composition of the antisense compounds and the assays in which they are being investigated. [0563] It is understood in the art that incorporation of nucleotide affinity modifications may allow for a greater number of mismatches compared to an unmodified compound. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences.
  • Tm melting temperature
  • Tm or ATm can be calculated by techniques that are familiar to one of ordinary skill in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex.
  • the single-stranded oligonucleotide can comprise a phosphorus-containing group at the 5'-end of a nucleotide sequence.
  • the 5'-end phosphorus-containing group can be 5'- end phosphate (5'-P), 5'-end phosphorothioate (5'-PS), 5'-end phosphorodithioate (5'-PS2), 5' -end vinylphosphonate (5' -VP), 5' -end methylphosphonate (MePhos), or 5'-deoxy-5'-C- malonyl ( ).
  • the 5'-VP can be either 5'-E-VP isomer (i.e., trans- vinylphosphate, ), 5'-Z-VP isomer (i.e., cis-vinylphosphate,
  • the single-stranded oligonucleotide comprises a phosphorus- containing group at the 5'-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the single-stranded oligonucleotide comprises a 5'-P in at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the single-stranded oligonucleotide comprises a 5' -PS in at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the single-stranded oligonucleotide comprises a 5' -VP in at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ). In one embodiment, the single-stranded oligonucleotide comprises a 5'-E-VP in at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ). In one embodiment, the single-stranded oligonucleotide comprises a 5'-Z-VP in at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the single-stranded oligonucleotide comprises a 5'-PS2 in at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • the single-stranded oligonucleotide comprises a 5'-deoxy-5'- C-malonyl in at least one nucleotide sequence (e.g., Z 1 and/or Z 2 ).
  • 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the single-stranded oligonucleotide is modified.
  • 50% of the single-stranded oligonucleotide is modified, 50% of all nucleotides present in the single-stranded oligonucleotide contain a modification as described herein.
  • each nucleotide of Z 1 and Z 2 of the single-stranded oligonucleotide is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2'- methoxyethyl, 2'- O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-O-N- methylacetamido ( 2'-O-NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O- aminopropyl (2'-O-AP), or 2'-ara-F.
  • the nucleotide sequence (e.g., Z 1 and/or Z 2 ) of the single- stranded oligonucleotide contains at least two different modifications.
  • the single-stranded oligonucleotide does not contain any 2'-F modification.
  • the single-stranded oligonucleotide comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages.
  • the single-stranded oligonucleotide comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages.
  • the two blocks of phosphorothioate or methylphosphonate intemucleotide linkages are separated by 16-18 phosphate intemucleotide linkages.
  • the nucleotide at position 1 of the 5 -'end of a nucleotide sequence is selected from the group consisting of A, dA, dU, U, and dT.
  • at least one of the first, second, and third base pair from the 5 '-end of the nucleotide sequence is an AU base pair.
  • the single-stranded oligonucleotide is 100% complementary to a target RNA to hybridize thereto and inhibits its expression through RNA interference. In another embodiment, the single-stranded oligonucleotide is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to a target RNA.
  • a single-stranded oligonucleotide capable of inhibiting the expression of a target gene.
  • the single-stranded oligonucleotide contains at least one thermally destabilizing nucleotide.
  • the thermally destabilizing nucleotide can occur, for example, between positions 14-17 of the 5 '-end of a nucleotide sequence (e.g., Z 1 and/or Z 2 ) of 21 nucleotides in length.
  • the nucleotide sequence can contain at least two modified nucleic acids that are smaller than a sterically demanding 2'-OMe modification.
  • the two modified nucleic acids that are smaller than a sterically demanding 2'-OMe are separated by 11 nucleotides in length.
  • the two modified nucleic acids are at positions 2 and 14 of the 5 e'nd.
  • the single-stranded oligonucleotide contains a sequence that can be represented by formula (II):
  • i and j are each independently 0 or 1; p and q are each independently 0-6; each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each Nb independently represents an oligonucleotide sequence comprising 1, 2, 3, 4, 5, or 6 modified nucleotides; each np and nq independently represent an overhang nucleotide; wherein Nb and Y do not have the same modification; wherein XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides.
  • the single-stranded oligonucleotide contains one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 2'-F modification(s). In one example, the single-stranded oligonucleotide contains nine or ten 2'-F modifications.
  • the single-stranded oligonucleotide may further comprise at least one phosphorothioate or methylphosphonate intemucleotide linkage.
  • the phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the single- stranded oligonucleotide.
  • the intemucleotide linkage modification may occur on every nucleotide on at least one nucleotide sequence; each intemucleotide linkage modification may occur in an alternating pattern on at least one nucleotide sequence.
  • the compound of the invention disclosed herein is a miRNA mimic.
  • miRNA mimics are double stranded molecules (e.g., with a duplex region of between about 16 and about 31 nucleotides in length) and contain one or more sequences that have identity with the mature strand of a given miRNA. Double- stranded miRNA mimics have designs similar to as described above for double-stranded iRNAs.
  • a miRNA mimic comprises a duplex region of between 16 and 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide), and all of the Cs and Us; the antisense strand modifications can comprise 2' F modification of all of the Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized intemucleotide linkages associated with a 2 nucleotide 3 ' overhang.
  • the compound of the invention disclosed herein is an antimir.
  • compound of the invention comprises at least two antimirs covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, for example a linker described in the disclosure, or non-covalently linked to each other.
  • antimir'' "microRNA inhibitor” or “miR inhibitor” are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the activity of specific miRNAs.
  • microRNA inhibitors comprise one or more sequences or portions of sequences that are complementary or partially complementary with the mature strand (or strands) of the miRNA to be targeted, in addition, the miRNA inhibitor can also comprise additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA.
  • the additional sequences can be the reverse complements of the sequences that are adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequences can be arbitrary sequences (having a mixture of A, G, C, U, or dT).
  • one or both of the additional sequences are arbitrary sequences capable of forming hairpins.
  • the sequence that is the reverse complement of the miRNA is flanked on the 5' side and on the 3' side by hairpin structures.
  • MicroRNA inhibitors when double stranded, can include mismatches between nucleotides on opposite strands. Furthermore, microRNA inhibitors can be linked to conjugate moieties in order to facilitate uptake of the inhibitor into a cell.
  • MicroRNA inhibitors including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13: 723-730 (2007) and in WO2007/095387 and WO 2008/036825 each of which is incorporated herein by reference in its entirety.
  • a person of ordinary skill in the art can select a sequence from the database for a desired miRNA and design an inhibitor useful for the methods disclosed herein.
  • compound of the invention disclosed herein is an antagomir.
  • the compound of the invention comprises at least two antagomirs covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, for example a linker described in the disclosure, or non-covalently linked to each other.
  • Antagomirs are RNA-like oligonucleotides that harbor various modifications for RNAse protection and pharmacologic properties, such as enhanced tissue and cellular uptake.
  • antagomir comprises a 2'-O-methyl modification at all nucleotides, a cholesterol moiety at 3 '-end, two phosphorothioate intersugar linkages at the first two positions at the 5 '-end and four phosphorothioate linkages at the 3 -'end of the molecule.
  • Antagomirs can be used to efficiently silence endogenous miRNAs by forming duplexes comprising the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing.
  • An example of antagomir-mediated miRNA silencing is the silencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which is expressly incorporated by reference herein in its entirety.
  • RNAa activating RNA
  • RNA activation by RNAa is long-lasting. Induction of gene expression has been seen to last for over ten days. The prolonged effect of RNAa could be attributed to epigenetic changes at dsRNA target sites.
  • the RNA activator can increase the expression of a gene. In some embodiments, increased gene expression inhibits viability, growth development, and/or reproduction.
  • compound of the invention disclosed herein is activating RNA.
  • the compound of the invention comprises at least two activating RNAs covalently linked to each other via a nucleotide-based or non- nucleotide-based linker, for example a linker described in the disclosure, or non-covalently linked to each other.
  • compound of the invention disclosed herein is a triplex forming oligonucleotide (TFO).
  • the compound of the invention comprises at least two TFOs covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, for example a linker described in the disclosure, or non- covalently linked to each other.
  • a nucleotide-based or non-nucleotide-based linker for example a linker described in the disclosure, or non- covalently linked to each other.
  • triplex forming oligonucleotides can be designed which can recognize and bind to polypurine/polypyrimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outline by Maher III, L.J., et al., Science (1989) vol.
  • oligonucleotides Modification of the oligonucleotides, such as the introduction of intercalators and intersugar linkage substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer, J Clin Invest 2003;l 12:487-94).
  • the triplex-forming oligonucleotide has the sequence correspondence: oligo 3'-A G G T duplex 5'-A G C T duplex 3'-T C G A [0591] However, it has been shown that the A- AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, Septl2, Epub). The same authors have demonstrated that TFOs designed according to the A- AT and G-GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific. [0592] Thus for any given sequence a triplex forming sequence can be devised. Triplex- forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 nucleotides.
  • TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both down- regulation and up-regulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94).
  • Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Pat. App. Nos. 2003 017068 and 2003 0096980 to Froehler et al, and 2002 0128218 and 2002 0123476 to Emanuele et al, and U.S. Pat. No.
  • the single-stranded oligonucleotide comprises at least one nucleic acid modification described herein.
  • such a modification can be present anywhere in the single-stranded oligonucleotide.
  • the modification can be present in one of the RNA molecules.
  • the naturally occurring base portion of a nucleoside is typically a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • a phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.
  • those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the naturally occurring linkage or backbone of RNA and of DNA is a 3' to 5' phosphodiester linkage.
  • nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U)
  • A purine nucleobase
  • G guanine
  • T cytosine
  • U uracil
  • modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein.
  • the unmodified or natural nucleobases can be modified or replaced to provide iRNAs having improved properties.
  • nuclease resistant oligonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the oligomer modifications described herein.
  • nucleobases e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine
  • substituted or modified analogs of any of the above bases and “universal bases'' can be employed.
  • the nucleotide is said to comprise a modified nucleobase and/or a nucleobase modification herein.
  • Modified nucleobase and/or nucleobase modifications also include natural, non-natural and universal bases, which comprise conjugated moieties, e.g. a ligand described herein.
  • Preferred conjugate moieties for conjugation with nucleobases include cationic amino groups which can be conjugated to the nucleobase via an appropriate alkyl, alkenyl or a linker with an amide linkage.
  • An oligomeric compound described herein can also include nucleobase (often referred to in the art simply as “base'') modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2- (alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)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,
  • a universal nucleobase is any nucleobase that can base pair with all of the four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the iRNA duplex.
  • 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-propyny
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in International Application No. PCT/US09/038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P.Ed.
  • a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5- methyl cytosine, or a G-clamp.
  • nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.
  • the single-stranded oligonucleotide provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomer, including a nucleoside or nucleotide, having a modified sugar moiety.
  • the furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid.
  • oligomeric compounds comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers that are LNA.
  • each of the linkers of the LNA compounds is, independently, — [C(R1)(R2)]n-, — [C(R1)(R2)]n-O— , — C(R1R2)-N(R1)-O— or — C(R1R2)-O — N(R1)-.
  • each of said linkers is, independently, 4'-CH 2 - 2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2 -O-2', 4'-(CH 2 ) 2 -O-2', 4'-CH 2 -O— N(R1)-2' and 4'-CH 2 - N(R1)-O-2'- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl.
  • LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring thereby forming a methyleneoxy (4'-CH 2 -O-2') linkage to form the bicyclic sugar moiety
  • 4'-CH 2 -O-2' linkage to form the bicyclic sugar moiety
  • the linkage can be a methylene ( — CH 2 -) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'-CH 2 -O-2') LNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ethyleneoxy (4'- CH 2 CH 2 -O-2') LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456: Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226).
  • Potent and nontoxic antisense oligonucleotides comprising BNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).
  • alpha-L-methyleneoxy (4'-CH 2 -O-2') LNA which has been shown to have superior stability against a 3 '-exonuclease.
  • the alpha-L-methyleneoxy (4'-CH 2 -O-2') LNA's were incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
  • Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance.
  • a representative list of preferred modified sugars includes but is not limited to bicyclic modified sugars, including methyleneoxy (4'-CH 2 -O-2') LNA and ethyleneoxy (4'- (CH 2 ) 2 -O-2' bridge) ENA; substituted sugars, especially 2'-substituted sugars having a 2'-F, 2'-OCH 3 or a 2'-O(CH 2 ) 2 -OCH 3 substituent group; and 4'-thio modified sugars. Sugars can also be replaced with sugar mimetic groups among others.
  • a modification at the 2' position can be present in the arabinose configuration
  • the term “arabinose configuration'' refers to the placement of a substituent on the C2' of ribose in the same configuration as the 2'-OH is in the arabinose.
  • the sugar can comprise two different modifications at the same carbon in the sugar, e.g., gem modification.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • an oligomeric compound can include one or more monomers containing e.g., arabinose, as the sugar.
  • the monomer can have an alpha linkage at the 1 ' position on the sugar, e.g., alpha-nucleosides.
  • the monomer can also have the opposite configuration at the 4' -position, e.g., C5' and H4' or substituents replacing them are interchanged with each other. When the C5' and H4' or substituents replacing them are interchanged with each other, the sugar is said to be modified at the 4' position.
  • the single- stranded oligonucleotide disclosed herein can also include abasic sugars, i.e., a sugar which lack a nucleobase at C-1' or has other chemical groups in place of a nucleobase at C1'. See for example U.S. Pat. No. 5,998,203, content of which is herein incorporated in its entirety. These abasic sugars can also be further containing modifications at one or more of the constituent sugar atoms.
  • the single-stranded oligonucleotide can also contain one or more sugars that are the L isomer, e.g. L-nucleosides. Modification to the sugar group can also include replacement of the 4'-0 with a sulfur, optionally substituted nitrogen or CH 2 group. In some embodiments, linkage between C1' and nucleobase is in a configuration.
  • Sugar modifications can also include acyclic nucleotides, wherein a C-C bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and/or at least one of ribose carbons or oxygen (e.g., C1', C2', C3', C4' or 04') are independently or in combination absent from the nucleotide.
  • a C-C bonds between ribose carbons e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4'
  • ribose carbons e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4'
  • acyclic nucleotide is wherein B is a modified or unmodified nucleobase, R 1 and R 2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • sugar modifications are selected from the group consisting of 2'-H, 2'-O-Me ( 2'-O-methyl), 2'-O-MOE ( 2'-O-methoxyethyl), 2'-F, 2'-O-[2- (methylamino)-2-oxoethyl] ( 2'-O-NMA), 2'-5-methyl, 2'-O-CH 2 -(4'-C) (LNA), 2'-O- CH 2 CH 2 -(4'-C) (ENA), 2'-O-aminopropyl ( 2'-O-AP), 2'-O-dimethylaminoethyl ( 2'-O- DMAOE), 2'-O-dimethylaminopropyl ( 2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'--
  • the hydrogen attached to C4' and/or C1' can be replaced by a straight- or branched- optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein backbone of the alkyl, alkenyl and alkynyl can contain one or more of O, S, S(O), SO2, N(R'), C(O), N(R')C(O)O, OC(O)N(R'), CH(Z'), phosphorous containing linkage, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic or optionally substituted cycloalkyl, where R' is hydrogen, acyl or optionally substituted aliphatic, Z' is selected from the group consisting of OR 11 , COR 11 , CO 2 R 11 , , NR 21 R 31 , CON NR 21 R, 31 CON(H) NR 21 R 31 ,
  • C4' and C5' together form an optionally substituted heterocyclic, preferably comprising at least one -PX(Y)-, wherein X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino, where M is independently for each occurrence an alkali metal or transition metal with an overall charge of +1; and Y is O, S, or NR', where R' is hydrogen, optionally substituted aliphatic.
  • this modification is at the 5 terminal of the iRNA.
  • LNA's include bicyclic nucleoside having the formula: wherein:
  • Bx is a heterocyclic base moiety
  • Ti is H or a hydroxyl protecting group
  • T2 is H, a hydroxyl protecting group or a reactive phosphorus group
  • Z is C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 1 -C 6 alkyl, substituted C 2 - C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, or substituted amide.
  • the compounds of the invention comprise at least one monomer of the formula: wherein
  • Bx is a heterocyclic base moiety
  • T 3 is H, a hydroxyl protecting group, a linked conjugate group or an intemucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound;
  • T 4 is H, a hydroxyl protecting group, a linked conjugate group or an intemucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; wherein at least one of T 3 and T 4 is an intemucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; and
  • Z is C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 1 -C 6 alkyl, substituted C 2 - C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, or substituted amide.
  • LNAs include, but are not limited to, (A) a-L- Methyleneoxy (4'-CH 2 -O-2') LNA, (B) ⁇ -D-Methyleneoxy (4'-CH 2 -O-2') LNA, (C) Ethyleneoxy (4'-(CH 2 ) 2 -O-2') LNA, (D) Aminooxy (4'-CH 2 -O — N(R)-2') LNA and (E) Oxyamino (4'-CH 2 -N(R) — O-2') LNA, as depicted below:
  • the single-stranded oligonucleotide comprises at least two regions of at least two contiguous monomers of the above formula. In certain embodiments, the single-stranded oligonucleotide comprises a gapped motif. In certain embodiments, the single- stranded oligonucleotide comprises at least one region of from about 8 to about 14 contiguous ⁇ -D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, the single- stranded oligonucleotide comprises at least one region of from about 9 to about 12 contiguous ⁇ -D-2'-deoxyribofuranosyl nucleosides.
  • the single-stranded oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (S)-cEt monomer of the formula: wherein Bx is heterocyclic base moiety.
  • monomers include sugar mimetics.
  • a mimetic is used in place of the sugar or sugar-intemucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
  • Representative examples of a sugar mimetics include, but are not limited to, cyclohexenyl or morpholino.
  • Representative examples of a mimetic for a sugar-intemucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances, a mimetic is used in place of the nucleobase.
  • nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.
  • linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming an oligomeric compound, e.g., an oligonucleotide.
  • Such linking groups are also referred to as intersugar linkage.
  • the two main classes of linking groups are defined by the presence or absence of a phosphorus atom.
  • Non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino ( — CH 2 -N(CH 3 )-O — CH 2 -), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ); siloxane ( — O — Si(H) 2 -O — ); and N,N'-dimethylhydrazine ( — CH 2 -N(CH 3 )-N(CH 3 )-).
  • Modified linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotides.
  • 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 linking group 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 linkage can be replaced by any of the following: S, Se, BR 3 (R is hydrogen, alkyl, aryl), C (i.e.
  • 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).
  • 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).
  • the phosphate linker can also be modified by replacement of bridging oxygen, (i.e. oxygen that links the phosphate to the sugar of the monomer), 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 monomer
  • 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.

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Abstract

Un aspect de la présente invention concerne un oligonucléotide simple brin présentant une séquence représentée par la formule (II) ou (III) : (5' - Z11 - 3')– L–QS–(5' - Z12 - 3') (II), (3' - Z11 - 5')– L–QS–(3' - Z12 - 5' (III), dans la formule (II) ou (III), Z11 est un premier oligonucléotide, comprenant 15 à 100 nucléotides éventuellement modifiés qui est sensiblement complémentaire à un gène cible ; Z12 est un second oligonucléotide, comprenant 10 à 100 nucléotides éventuellement modifiés qui est sensiblement complémentaire à Z11 ; Z11 et Z12 <sb /> permettent de former une région duplexée intra-brin comprenant au moins 7 paires de bases consécutives ; QS représente 0 à 12 nucléotides éventuellement modifiés ; L est un groupe de liaison facultatif ; au moins un nucléotide dans la formule (II) est un nucléotide modifié ; et au moins un nucléotide dans la formule (III) est un nucléotide modifié, au moins un nucléotide à l'extrémité 3' de Z11, pour la formule (II), ou au moins un nucléotide à l'extrémité 5' de Z11, pour la formule (III), dans l'un ou l'autre cas conjointement avec L et QS, formant une région de boucle reliant Z11 et Z12.
EP24731742.3A 2023-05-12 2024-05-10 Oligonucléotides à boucle simple brin Pending EP4709855A2 (fr)

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