WO2023283434A1 - Aminooxy click chemistry (aocc): a versatile bioconjugation approach - Google Patents

Aminooxy click chemistry (aocc): a versatile bioconjugation approach Download PDF

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WO2023283434A1
WO2023283434A1 PCT/US2022/036538 US2022036538W WO2023283434A1 WO 2023283434 A1 WO2023283434 A1 WO 2023283434A1 US 2022036538 W US2022036538 W US 2022036538W WO 2023283434 A1 WO2023283434 A1 WO 2023283434A1
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optionally substituted
alkyl
compound
group
solid support
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WO2023283434A9 (en
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Muthiah Manoharan
Shohei Mori
Dhrubajyoti Datta
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Alnylam Pharmaceuticals Inc
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Alnylam Pharmaceuticals Inc
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Priority to EP22838475.6A priority Critical patent/EP4367125A4/en
Priority to US18/576,561 priority patent/US20250304613A1/en
Priority to JP2024500191A priority patent/JP2024525552A/ja
Publication of WO2023283434A1 publication Critical patent/WO2023283434A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • 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

Definitions

  • the present disclosure relates generally to monomers and methods for conjugating one or more ligands to oligonucleotides.
  • m1 is 0, 1, 2 or 3 (e.g., m1 is 0 or 1); J is O, S, CH 2 or N-alkyl (e.g., NCH 3 ); R 1 is B or –B-L 1 -R 6 ; L 1 is a linker; B is a nucleobase; R 2 is hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 1- 30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2- 30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxycarboxylate,
  • a compound selected from the group consisting of formulae IIa-IIg where: wherein: J is O, S, CH 2 or N-alkyl (e.g., NCH 3 ); L 3 is a linker; R 3NN is independently hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino,
  • the compound is of Formula Ia.
  • the compunds of Formulae Ia-Ig, Ii and IIa-IIg are useful in the synthesis oligonucleotides. Accordingly, in another aspect, provided herein is an oligonucleotide prepared using a compound selected from compounds of formulae Ia-Ig, Ii and IIa-IIf.
  • an oligonucleotide comprising at least one nucleoside selected from the group consisting of nucleotides of formulae IIIa-IIIh and IVa-IVg, where: [0008] In nucleoside of formulae IIIa-IIIh and IVa-IVg, m1 is 0, 1, 2 or 3 (e.g., m1 is 0 or 1); J is O, S, CH 2 or N-alkyl (e.g., NCH 3 ); R 1 is B or –B-L 1 -R 6 ; L 1 is a linker; L 3 is a linker; B is a nucleobase; R 32 is hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 1 - 30haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2 - 3 0 alkynyl, optionally substituted
  • one of R 32 or R 33 is a bond to an internucleotide linkage to a subsequent nucleotide or R 35 is a bond to an internucleotide linkage to a preceding nucleotide.
  • R 35 is a bond to an internucleotide linkage to a preceding nucleotide.
  • a double-stranded nucleic acid comprising a first strand and a second strand complementary to the first strand, and wherein at least one of the first and second strand is an oligonucleotide comprising a nucleotide of selected from Formulae IIIa-IIIg and IVa-IVg described herein.
  • a method for inhibiting or reducing the expression of a target gene in a subject comprises administering to the subject: (i) a double- stranded RNA described herein, wherein one of the strands of the dsRNA is complementary to a target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to a target gene.
  • a double- stranded RNA described herein wherein one of the strands of the dsRNA is complementary to a target gene
  • an oligonucleotide described herein wherein the oligonucleotide is complementary to a target gene.
  • FIG.1 is a schematic representation of some exemplary embodiments of the disclosure.
  • FIGS.2A-6B depict exemplary nucleotide monomers according to embodiments of the disclosure.
  • FIGS. 6C and 6D depic exemplary nucleotide monomers according to embodiments of the disclosure as incorporated into nucleic acids.
  • FIGS. 7-10 depict exemplary non-nucleotide monomers based on prolinol scaffods (FIG.7), serinol scaffods (FIG.8), D- and L-Threoninol scaffolds (FIG.9), and conjugates derived from Pentaerythritol and Norbornyl scaffolds (FIG. 10) according to embodiments of the disclosure.
  • FIG.11 depicts exemplary ligands with aldehyde and ketone linkers.
  • FIGS.12-14 are schematic respresentations of exemplary modification sites in double- stranded nuclei acids. [0019] FIGS.
  • FIG.16 depicts synthesis of AOCC modified oligonucleotides from AOCC-conjugate building blocks according to an embodiment of the disclosure.
  • FIGS. 17A-17D show analysis of oligonucleotide conjugated according to embodiments of the disclosure.
  • FIG.18 is a schematic representation of some exemplary aspects of the disclosure.
  • FIG. 19 depicts some exemplary AOCC building blocks according to some embodiments of the disclosure.
  • FIG.20 is a schematic representation of some exemplary aspects of the disclosure. [0025] FIGS.
  • FIG. 21-24 depict exemplary building blocks according to embodiments of the disclosure.
  • FIG.25 is a scheme showing oligonucleotides synthesis from AOCC building blocks.
  • FIG.26 depicts post-synthetic conjugation of oligonucleotides.
  • FIG.27 shows LCMS analysis of crude conjugaties prepared from poly-dT sequence.
  • FIG.28 depicts post-synthetic conjugation of oligonucleotides on solid support.
  • FIG.29 shows LCMS analysis of crude conjugates from post-synthetic conjugation of oligonucleotides on solid support.
  • FIG.30 depicts some exemplary peptides for AOCC chemistry.
  • FIG.31 depicts some ecemplary non-nucleotide scaffolds for AOCC.
  • FIG.32 depicts some exemplary carbonyl and acid compounds for AOCC modifications.
  • FIG. 33 depicts some exemplary compounds comprising a 2’- or 3’-modified sugar according to some embodiments of the disclsoure.
  • FIG. 34 depicts some exemplary compounds comprising a C4- or C5-substitued pyrimidine according to some embodiments of the disclosure.
  • FIG. 35 depicts some exemplary compounds comprising a N6 or C8-substitued pyrimidine according to some embodiments of the disclosure.
  • FIGS. 37A and 37B depict structures of some abbreviations used in the nucleic acid sequences disclosed herein.
  • FIGS.38A and 38B depict some exemplary embodiments of the disclosure.
  • DETAILED DESCRIPTION It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
  • the use of the singular includes the plural unless specifically stated otherwise.
  • the use of “or” means “and/or” unless stated otherwise.
  • R 1 (nucleobase)
  • R 1 can be a nucleobase or a nucleobase comprising a –L 1 -R 6 group.
  • the nucleobase can be a natural nucleobase or a non-natural nucleobase.
  • a “non-natural nucleobase” is meant a nucleobase other than adenine, guanine, cytosine, uracil, or thymine.
  • non-natural nucleobases include, but are not limited to, inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substitute
  • purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, content of all which is incorporated herein by reference.
  • the non-natural nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8- (hydroxyl)adenine, 8-(thioalkyl)adenine, 8-(thiol)adenine, N 6 -(isopen
  • a non-natural nucleobase is a modified nucleobase, i.e., the nucleobase comprises a nucleobase modification described herein, e.g., the nucleobase is a substituted or modified analog of any of the natural nucleobases.
  • nucleobase modifications include, but not limited to: C-5 pyrimidine with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities, N 2 - and N 6 - with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities of purines, G-clamps, guanidinium G-clamps, and pseudouridine known in the art.
  • the non-natural nucleobase is a universal nucleobase.
  • a universal nucleobase is any modified or unmodified natural or non-natural nucleobase that can base pair with all of adenine, cytosine, guanine and uracil without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide comprising the universal nucleobase.
  • Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza- 7-deazaadenine, 4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6- methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4- methylinolyl, 4,6-dimethylindolyl, phenyl, nap
  • the non-matural nucleobase is a protected nucleobase.
  • a “protected nucleobase” referes to a nucleobase comprising a nitrogen protecting group, and/or an oxygen protecting group, and/or a sulfur protecting group.
  • the non-natural nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.
  • R 1 is a pyrimidine nucleobase comprising –L 1 -R 6 .
  • R 1 is a pyrimidine nucleobase comprising –L 1 -R 6 at the C5 position.
  • R 1 is uracil substituted with –L 1 -R 6 at the C5 position.
  • R 1 is cytosine substituted with –L 1 -R 6 at the C5 position.
  • R 1 is 4-aminopyrimidine nucleobase comprising –L 1 -R 6 linked to the 4-amino group.
  • R 1 is cytidine comprings –L 1 -R 6 linked to the amino at the C4 position.
  • R 1 is a purine nucleobase comprising –L 1 -R 6 .
  • R 1 is a purine nucleobase comprising –L 1 -R 6 at the C2, N6, or C8 position.
  • R 1 is adenine substited with –L 1 -R 6 at one of C2, N6, or C8 position.
  • R 1 is a 2-amino purine nucleobase comprising –L 1 -R 6 .
  • R 1 is a 2-aminopurine nucleobase comprising –L 1 - R 6 at the N2, N6, or C8 position.
  • R 1 is guanine substited with –L 1 -R 6 at one of N2, N6, or C8 position.
  • R 1 is a N7-deaza purine nucleobase comprising –L 1 -R 6 .
  • R 1 is a N7-deaza purine nucleobase comprising –L 1 -R 6 at the C2, N6, C8 or N7-deaza position.
  • R 1 is a N7- deazaadenine subtittuted with –L 1 -R 6 at one of C2, N6, C8 or N7-deaza position.
  • R 1 is a 2-amino- N7- deaza purine nucleobase comprising –L 1 -R 6 .
  • R 1 is a 2-amino-N7-deaza purine nucleobase comprising –L 1 -R 6 at the N2, N6, C8 or N7-deaza position.
  • R 1 is a N7-deazaguanine substituted with –L 1 -R 6 at one of C2, N6, C8 or N7-deaza position.
  • L 1 is a linker.
  • linker means an organic moiety that connects two parts of a compound.
  • Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR N1 , C(O), C(O)O, C(O)NR 1 , SO, SO 2 , SO 2 NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylaryl
  • the linker is a cleavable linker.
  • Cleavable linkers are those that rely on processes inside a target cell to liberate the two parts the linker is holding together, as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by specific enzymes (e.g. proteases) within the cell.
  • cleavable linkers allow the two parts to be released in their original form after internalization and processing inside a target cell.
  • Cleavable linkers include, but are not limited to, those whose bonds can be cleaved by enzymes (e.g., peptide linkers); reducing conditions (e.g., disulfide linkers); or acidic conditions (e.g., hydrazones and carbonates).
  • enzymes e.g., peptide linkers
  • reducing conditions e.g., disulfide linkers
  • acidic conditions e.g., hydrazones and carbonates.
  • the cleavable linker comprises at least one cleavable linking group.
  • a cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together.
  • the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood or serum of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood.
  • degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
  • redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g
  • a cleavable linkage group such as a disulfide bond can be susceptible to pH.
  • the pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1- 7.3.
  • Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0.
  • Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
  • a linker can include a cleavable linking group that is cleavable by a particular enzyme.
  • the type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
  • the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • a degradative agent or condition
  • the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
  • the evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals.
  • useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
  • cleavable linking groups are redox cleavable linking groups, which may be used in the dsRNA molecule according to the present invention that are cleaved upon reduction or oxidation.
  • An example of reductively cleavable linking group is a disulfide linking group (-S-S-).
  • a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein.
  • a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell.
  • the candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions.
  • candidate compounds are cleaved by at most 10% in the blood.
  • useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).
  • the rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
  • Phosphate-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by agents that degrade or hydrolyze the phosphate group.
  • agents that degrade or hydrolyze the phosphate group are enzymes such as phosphatases in cells.
  • phosphate-based linking groups are -0-P(0)(0Rk)-0-, -O- P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O- P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(S)(
  • Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S- P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(O)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-, -O-P(S)(H)-S-.
  • Acid cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are linking groups that are cleaved under acidic conditions.
  • acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid.
  • specific low pH organelles such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups.
  • Acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids.
  • a preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.
  • Ester-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as esterases and amidases in cells.
  • ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups.
  • Ester cleavable linking groups have the general formula - C(O)O-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.
  • Peptide-based cleavable linking groups which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as peptidases and proteases in cells.
  • Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.
  • Peptide-based cleavable groups do not include the amide group (-C(O)NH-).
  • the amide group can be formed between any alkylene, alkenylene or alkynylene.
  • a peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins.
  • the peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group.
  • Peptide-based cleavable linking groups have the general formula – NHCHR A C(O)NHCHR B C(O)-, where R A and R B are the R groups of the two adjacent amino acids.
  • L 1 is an optionally subtitued C 1 -C 20 alkylene, (e.g., –(CH 2 )b–, where b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 19 or 20), or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • L 1 is an optionally substituted subtitued C10- C14alkylene or an optionally substituted C2-C6alkynylene.
  • L 1 is an optionally substituted C2alkylene, C12alkylene or C3alkynylene.
  • L 1 is an optionally substitute C2-C16alkylene.
  • L 1 is –C(O)NH-alipahtic or –aliphatic-C(O)NH-alipahtic.
  • L 1 is –(CH 2 ) nl –C(O)NH-(CH 2 ) nl -CH 2 – or –CH 2 CH 2 C(O)NH-(CH 2 ) nl -CH 2 –, where each nl is independently 0-16.
  • L 1 is –C(O)NH-(CH 2 ) nl -CH 2 –.
  • L 1 is –CH 2 CH 2 C(O)NH-(CH 2 ) nl -CH 2 –.
  • each Z is independently absent, a bond, O, S, or NR NR6 , where R NR6 is independently H, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted hterocyclyl, or a nitrogen protecting group.
  • Z is O.
  • Z is S.
  • Z is NR NR6 .
  • R 2 is –Z-L 2 -R 6 , hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O)rCH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH)sCH
  • R 322 can be H, hydroxyl protecting group, optionally substituted C 1-30 alkyl, C 1 - 30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 323 can be H, sulfur protecting group, optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 324 can be H, hydroxyl protecting group, optionally substituted C 1-30 alkyl, C 1 - 30haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl.
  • R 325 can be hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.
  • R 326 can be can be hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1 - 30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.
  • R 2 is –Z-L 2 -R 6 , hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O)rCH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 325 , NHC(O)R 324 .
  • R 2 is–Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ).
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxy
  • R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido
  • alkoxyalkylamine e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido
  • alkoxyalkylamine e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmeth
  • R 2 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, or alkoxyalkyl (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido).
  • R 2 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O- DMAEOE) or -O-N-methylacetamido (-O-NMA).
  • R 2 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O- DMAEOE) or -O-N-methylacetamido (-O-NMA).
  • R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O-DMAEOE) or - O-N-methylacetamido (-O-NMA).
  • R 2 is –Z-L 2 -R 6 .
  • R 2 is – Z-L 2 -R 6 , where Z is O.
  • R 2 is –Z-L 2 -R 6 .
  • R 2 is – Z-L 2 -R 6 , where L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R 2 is –Z-L 2 -R 6 , where L 2 is a polyethylene glycol (PEG).
  • R 2 is –Z-L 2 -R 6 , where Z is O and L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substitute
  • R 2 is –Z-L 2 -R 6 , where Z is O and L 2 is a polyethylene glycol (PEG).
  • R 2 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ .
  • R 2 is –Z-L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1 - 30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl
  • a ligand e.g., a
  • R 2 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 2 and R 4 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y-C(R 10 R 11 ) v -2’; v is 1, 2 or 3; where Y is -O-, -CH 2 -, - CH(Me)-, -C(CH 3 ) 2 -, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, - N(R 12 )C(O)-, or -C(O)N(R 12 )-; R 10 and R 11 independently are H, optionally substituted C 1 -C 6 alkyl, optionally substituted C2-C6alkenyl or optionally substituted C2-C6alkynyl; R
  • v is 1. In some other embodiments of any one of the aspects, v is 2. In some embodiments, Y is O. [0099] In some embodiments, R 2 and R 4 taken together are 4’-C(R 10 R 11 ) v -O-2’. [0100] It is noted that R 10 and R 11 attached to the same carbon can be same or different. For example, one of R 10 and R 11 can be H and the other of the R 10 and R 11 can be an optionally substituted C 1 -C 6 alkyl.
  • R 10 and R 11 independently are H or C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • one of R 10 and R 11 is H and the other is C 1 -C 6 alkyl, optionally substituted with a C 1 -C 6 alkoxy.
  • one of R 10 and R 11 is H and the other is –CH 3 or CH 2 OCH 3 .
  • R 10 and R 11 attached to the same C are the same.
  • R 10 and R 11 attached to the same C are H.
  • R 2 and R 4 taken together are 4’-CH 2 - O-2’, 4’-CH(CH 3 )-O-2’, 4’-CH(CH 2 OCH 3 )-O-2’, or 4’- CH 2 CH 2 -O-2’.
  • R 2 and R 4 taken together are 4’- CH 2 CH 2 -O-2’.
  • R 2 and R 4 taken together are 4’-C(R 10 R 11 )-O-2’, and where one of R 10 and R 11 is H and other is alkyl, secondary alkyl, homo-branched alkyl, hetero-branched alkyl, alkyl carboxylic esters, alkyl amines, or alkyl ether.
  • R 2 and R 4 taken together are 4’-CHR 11 -O-2’, where R 11 is alkyl, secondary alkyl, homo-branched alkyl, hetero-branched alkyl, alkyl carboxylic esters, alkyl amines, or alkyl ether.
  • R 2 is a reactive phosphorus group.
  • reactive phosphorus groups are useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages.
  • Such reactive phosphorus groups are known in the art and contain phosphorus atoms in P III or P V valence state including, but not limited to, phosphoramidite, H- phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries.
  • Reactive phosphorous group in the form of phosphoramidites (P III chemistry) as reactive phosphites are a preferred reactive phosphorous group for solid phase oligonucleotide synthesis.
  • the intermediate phosphite compounds are subsequently oxidized to the Pv state using known methods to yield phosphodiester or phosphorothioate internucleoside linkages.
  • the reactive phosphorous group is -OP(OR P )(N(R P2 ) 2 ), -OP(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )(N(R P2 ) 2 ), - OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, - OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 .
  • R P is an optionally substituted C 1 - 6 alkyl.
  • R p is a C 1-6 alkyl, optionally substituted with a CN or –SC(O)Ph.
  • R p is cyanoethyl (-CH 2 CH 2 CN).
  • each R P2 is independently optionally substituted C 1-6 alkyl.
  • each R P2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl. It is noted that when two or more R P2 groups are present in the reactive phosphorous group, they can be same or different.
  • each R P2 is isopropyl.
  • both R P2 taken together with the nitrogen atom to which they are attached form an optionally substituted 3-8 membered heterocyclyl.
  • R P and one of R P2 taken together with the atoms to which they are attached form an optionally substituted 4-8 membered heterocyclyl.
  • each R P3 is independently optionally substituted C 1 -6alkyl.
  • R P3 is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 - C 6 alkoxy.
  • the reactive phosphorous group is - OP(OR P )(N(R P2 ) 2 ).
  • the reactive phosphorous group is -OP(OR P )(N(R P2 ) 2 ), where R P is cyanoethyl (-CH 2 CH 2 CN) and each R P2 is isopropyl.
  • R 2 is - OP(OR P )(N(R P2 ) 2 ), -OP(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )(N(R P2 ) 2 ), - OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, - OP(O)(OR P )R P3 , -OP(S)(OR P )
  • R 2 is -OP(OR P ) (N(R P2 ) 2 ), - OP(SR P )(N(R P2 ) 2 ), -OP(O)(OR P )(N(R P2 ) 2 ), -OP(S)(OR P )(N(R P2 ) 2 ), -OP(O)(SR P )(N(R P2 ) 2 ), - OP(O)(OR P )H, -OP(S)(OR P ) an optionally substituted C 1-6 alkyl, each R P2 is independently optionally substituted C 1-6 alkyl; and each R P3 is independently optionally substituted C 1-6 alkyl.
  • R 2 is -OP(OR P )(N(R P2 ) 2 ).
  • the R 2 is -OP(OR P )(N(R P2 ) 2 ), where R P is cyanoethyl (-CH 2 CH 2 CN) and each R P2 is isopropyl.
  • R 2 is a solid support or a linker covalently attached to a solid support.
  • R 2 is –OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • R 2 is –OC(O)CH 2 CH 2 CO 2 H.
  • R 322 when R 2 is –OR 322 , R 322 can be hydrogen or a hydroxyl protecting group. [0117] When R 2 is –SR 323 , R 323 can be hydrogen or a sulfur protecting group. Accordingly, in some embodiments of any one of the aspects, R 323 is hydrogen.
  • R 2 is -O(CH 2 CH 2 O)rCH 2 CH 2 OR 324
  • r can be 1-50
  • R 324 is independently for each occurrence H, C 1 -C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 325
  • R 325 is independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 2 is -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 325
  • s can be 1-50 and R 325 can be independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 2 is hydrogen, halogen, –OR 322 , or optionally substituted C 1 -C 30 alkoxy.
  • R 2 is halogen, –OR 322 , or optionally substituted C 1 -C 30 alkoxy.
  • R 2 is F, OH or optionally substituted C 1 -C 30 alkoxy.
  • R 2 is C 1 -C 30 alkoxy optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 2 is –O(CH 2 ) t CH 3 , where t is 1-21.
  • t is 14, 15, 16, 17 or 18. In one non-limiting example, t is 16.
  • R 2 is –O(CH 2 ) u R 327 , where u is 2-10; R 327 is C 1 -C 6 alkoxy, amino (NH 2 ), CO 2 H, OH or halo.
  • R 327 is -CH 3 or NH 2 .
  • R 2 is –O(CH 2 ) u - OMe or R 2 is –O(CH 2 ) u NH 2 .
  • u is 2, 3, 4, 5 or 6.
  • u is 2, 3 or 6.
  • u is 2.
  • R 2 is a C 1 - C6haloalkyl.
  • R 2 is a C 1 -C 4 haloalkyl.
  • R 2 is –CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 or -CF 2 (CF3) 2 .
  • R 2 is – OCH(CH 2 OR 328 )CH 2 OR 329 , where R 328 and R 329 independently are H, optionally substituted C 1 - C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 328 and R 329 independently are optionally substituted C 1 -C 30 alkyl.
  • R 2 is – CH 2 C(O)NHR 3210 , where R 3210 is H, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 - C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 3210 is H or optionally substituted C 1 -C 30 alkyl.
  • R 3210 is optionally substituted C 1 -C 6 alkyl.
  • R 3 is -Z-L 2 -R 6 , hydrogen, halogen, -OR 332 , -SR 333 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O)rCH 2 CH 2 OR 334 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 335 , NHC(O)R 336
  • R 3 is –Z-L 2 -R 6 , hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH)sCH 2 CH 2 -R 325 , NHC(O)R 324 .
  • R 3 is–Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ).
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxy
  • R 3 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido
  • alkoxyalkylamine e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido
  • alkoxyalkylamine e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmeth
  • R 3 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, or alkoxyalkyl (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido).
  • R 3 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O-DMAEOE) or -O-N-methylacetamido (-O-NMA).
  • R 3 is hydrogen, hydroxyl, protected hydroxyl or methoxy.
  • R 3 is –Z-L 2 -R 6 .
  • R 3 is – Z-L 2 -R 6 , where Z is O.
  • R 3 is –Z-L 2 -R 6 .
  • R 3 is – Z-L 2 -R 6 , where L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R 3 is –Z-L 2 -R 6 , where L 2 is a polyethylene glycol (PEG).
  • R 3 is –Z-L 2 -R 6 , where Z is O and L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substitute
  • R 3 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ .
  • R 3 is –Z-L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1 - 30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl
  • a ligand e.g., a
  • R 3 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 3 is a reactive phosphorous group.
  • only one of R 2 and R 3 is a reactive phosphorous group.
  • R 3 is a solid support or a linker covalently attached to a solid support.
  • R 3 is –OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • Z is a solid support.
  • R 332 can be hydrogen or a hydroxyl protecting group.
  • R 332 can be hydrogen in some embodiments of any one of the aspects described herein.
  • R 3 is – OC(O)CH 2 CH 2 CO 2 H.
  • R 333 can be hydrogen or a sulfur protecting group. Accordingly, in some embodiments of any one of the aspects, R 333 is hydrogen.
  • R 3 is -O(CH 2 CH 2 O) r CH 2 CH 2 OR 334 , r can be 1-50; R 334 is independently for each occurrence H, C 1 -C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 335 ; and R 335 is independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 3 is -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 335 , s can be 1-50 and R 335 can be independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 3 is hydrogen, halogen, –OR 332 , or optionally substituted C 1 -C 30 alkoxy.
  • R 3 is halogen, –OR 332 , or optionally substituted C 1 -C 30 alkoxy.
  • R 3 is F, OH or optionally substituted C 1 -C 30 alkoxy.
  • R 3 is C 1 -C 30 alkoxy optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 3 is –O(CH 2 ) t CH 3 , where t is 1-21.
  • R 3 is –O(CH 2 ) u R 337 , where u is 2-10; R 337 is C 1 -C 6 alkoxy, amino (NH 2 ), CO 2 H, OH or halo.
  • R 337 is -CH 3 or NH 2 .
  • R 3 is –O(CH 2 ) u - OMe or R 3 is –O(CH 2 ) u NH 2.
  • R 3 is a C 1 - C6haloalkyl.
  • R 3 is a C 1 -C 4 haloalkyl.
  • R 3 is –CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 or -CF 2 (CF 3 ) 2 .
  • R 3 is – OCH(CH 2 OR 338 )CH 2 OR 339 , where R 338 and R 339 independently are H, optionally substituted C 1 - C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 338 and R 339 independently are optionally substituted C 1 -C 30 alkyl.
  • R 3 is – CH 2 C(O)NHR 3310 , where R 3310 is H, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 - C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 3 and R 4 taken together with the atoms to which they are attached form an optionally substituted C 3-8 cycloalkyl, optionally substituted C 3-8 cycloalkenyl, or optionally substituted 3-8 membered heterocyclyl.
  • R 4 can be –Z-L 2 -R 6 , hydrogen, optionally substituted C 1 -6alkyl, optionally substituted C2-6alkenyl, optionally substituted C2-6alkynyl, or optionally substituted C 1 -6alkoxy.
  • R 4 can be hydrogen, optionally substituted C 1 -6alkyl or optionally substituted C 1 -6alkoxy.
  • R 4 is –Z-L 2 -R 6 .
  • R 4 is – Z-L 2 -R 6 , where Z is a bond.
  • R 4 is –L 2 -R 6 .
  • R 4 is –L 2 -R 6 .
  • L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R 4 is –L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ .
  • R 3 is –L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted hetero
  • R 3 is –L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 4 is H.
  • R 5 is R 6 , -Z-L 2 -R 6 , R 551 , hydrogen, hydroxyl, optionally substituted C 1 -6alkyl-R 551 , optionally substituted -C2-6alkenyl-R 551 , or optionally substituted -C2-6alkynyl-R 551 , where R 551 can be –OR 552 , -SR 553 , hydrogen, a phosphorous group, a solid support or a linker to a solid support. When R 551 is –OR 552 , R 552 can be H or a hydroxyl protecting group.
  • R 551 when R 551 is –SR 553 , R 553 can be H or a sulfur protecting group.
  • R 5 is R 6 .
  • R 5 is -O-N(R 7 )R 7’ .
  • R 5 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, polyethylene glycols (PEGs), nitrogen protecting group.
  • a ligand e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins,
  • R 5 is -O-N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 5 is –Z-L 2 -R 6 .
  • R 5 is – Z-L 2 -R 6 , where Z is O.
  • R 5 is –Z-L 2 -R 6 .
  • R 5 is – Z-L 2 -R 6 , where L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R 5 is –Z-L 2 -R 6 , where L 2 is a polyethylene glycol (PEG).
  • R 5 is –Z-L 2 -R 6 , where Z is O and L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substitute
  • R 5 is –Z-L 2 -R 6 , where Z is O and L 2 is a polyethylene glycol (PEG).
  • R 5 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ .
  • R 5 is –Z-L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1- 30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optional
  • R 5 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 5 is –OR 552 or - SR 553 .
  • R 552 is a hydroxyl protecting group.
  • exemplary hydroxyl protecting groups for R 552 include, but are not limited to, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4′-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9- yl (MOX).
  • DMT 4,4′-dimethoxytrityl
  • Pixyl 9-phenylxanthine-9-yl
  • MOX 9-(p-methoxyphenyl)xanthine-9- yl
  • R 5 is –OR 552 and R 552 is 4,4′-dimethoxytrityl (DMT), e.g., R 5 is –O-DMT.
  • R 5 is –CH(R 554 )-R 551 , where R 554 is hydrogen, halogen, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 - C 30 alkenyl, optionally substituted C 2 -C 30 alkynyl, or optionally substituted C 1 -C 30 alkoxy.
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 551 is a reactive phosphorous group.
  • At least one at least one R 555 in P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ) is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2- 30 alkynyl, or an oxygen-protecting group.
  • At least one R 555 is H and at least one R 555 is other than H in -P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ).
  • all R 555 are H in -P(O)(OR 555 ) 2 , - P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), - OP(S)(SR 556 ) 2 , -SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 555 are other than H in in - P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , -OP(S)(OR 555 ) 2 , - OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is H.
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is other than H.
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl, or an sulfur-protecting group.
  • At least one R 556 is H and at least one R 556 is other than H in -P(S)(SR 556 ) 2 , -OP(S)(SR 556 ) 2 and -SP(S)(SR 556 ) 2 .
  • all R 556 are H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 556 are other than H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • R 3 is a reactive phosphorous group, a solid support, a linker to a solid support, and R 5 is a protected hydroxyl.
  • R 2 is a reactive phosphorous group, a solid support, a linker to a solid support
  • R 5 is a protected hydroxyl.
  • R 5 is R 6
  • one of R 2 and R 3 is a reactive phosphorous group, a solid support, a linker to a solid support.
  • R 6 is -O-N(R 7 )R 7’ .
  • R 6 N-OR 7 ,.
  • R 6 is -N(R 7 )-OR 7’ or –or –N(R 7’ )-OR 7 .
  • R 7 and R 7’ [0207] Embodiments of the various aspects described herein include the groups R 7 and R 7’ .
  • Each R 7 and R 7’ can be independently selected from the groups consisting of H, carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucelsides and nucleotides, oligonucleotides, detectable labels, diagnostic agents (e.g., bitoin), fluorescent dyes, polyethylene glycols (PEGs), antibodies, antibody fragments (e.g., nanobodies).
  • diagnostic agents e.g., bitoin
  • fluorescent dyes e.g., polyethylene glycols (PEGs)
  • PEGs polyethylene glycols
  • antibodies e.g., nanobodies
  • ligands modify one or more properties of the attached molecule (e.g., the oligonucleotide described herein) including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Ligands are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound.
  • a preferred list of ligands includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • Preferred ligands amenable to the present invention include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • cholic acid Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053
  • a thioether e.g., hexyl-S-tritylthiol
  • a thiocholesterol (Oberhauser 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-hydroxylpropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2 , polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine
  • porphyrins e.g., TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g., EDTA
  • lipophilic molecules e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1- pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytr
  • 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 ⁇ 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.
  • Exemplary amphipathic peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S.
  • endosomolytic ligand refers to molecules having endosomolytic properties.
  • Endosomolytic ligands promote the lysis of and/or transport of the composition of the invention, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell.
  • Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and brached 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, AALEALAEALEALAEALEALAEAAAAGGC (GALA); AALAEALAEALAEALAEALAAAAGGC (EALA); ALEALAEALEALAEA; GLFEAIEGFIENGWEGMIWDYG (INF-7); GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2); GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3); GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (GALA); GL
  • 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-die
  • Exemplary cell permeation peptides include, but are not limited to, RQIKIWFQNRRMKWKK (penetratin); GRKKRRQRRRPPQC (Tat fragment 48-60); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence based peptide); LLIILRRRIRKQAHAHSK (PVEC); GWTLNSAGYLLKINLKALAALAKKIL (transportan); KLALKLALKALKAALKLA (amphiphilic model peptide); RRRRRRRRR (Arg9); KFFKFFKFFK (Bacterial cell wall permeating peptide); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1); ACYCRIPACIAGERRYGTCIYQGRLWAFCC ( ⁇ -defensin); DHYNCVSSGGQCLYSACPIFTKIQGTCY
  • 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. GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacids and lectins.
  • the term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.
  • PK modulating ligand and “PK modulator” refers to molecules which can modulate the pharmacokinetics of oligonucleotides described herein.
  • Some exemplary PK modulator include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, and transthyretia-binding ligands (e.g., tetraiidothyroacetic acid, 2, 4, 6-triiodophenol and flufenamic acid).
  • lipophilic molecules bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, car
  • Oligomeric compounds that comprise a number of phosphorothioate intersugar linkages are also known to bind to serum protein, thus short oligomeric compounds, e.g. oligonucleotides of comprising from about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), and that comprise a plurality of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).
  • ligands e.g. as PK modulating ligands
  • the PK modulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and/or phosphorodithioate linkages. In some embodiments, all internucleoside linkages in PK modulating oligonucleotide are phosphorothioate and/or phosphorodithioates linkages.
  • aptamers that bind serum components e.g. serum proteins
  • Binding to serum components e.g.
  • the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties.
  • a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties.
  • all the ligands have different properties.
  • the ligand has a structure shown in any of Formula (IV) – (VII): ; wherein: q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , T 5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH 2 , CH 2 NH or CH 2 O; Q 2A , Q 2B , Q 2B , q 3A , q
  • the ligand is of Formula (VII): , wherein L 5A , L 5B and L 5C represent a monosaccharide, such as GalNAc derivative.
  • Exemplary ligands include, but are not limited to, the following:
  • the ligand is a ligand described in US Patent No. 5,994,517 or US Patent No. 6,906,182, content of each of which is incorporated herein by reference in its entirety.
  • the ligand can be a tri-antennary ligand described in Figure 3 of US Patent No. 6,906,182.
  • the ligand is selected from the following tri-antennary ligands:
  • R L is a ligand
  • ligands when more than one ligands are present, they can be same or different. Accordingly, in some embodiments of any one of the aspects described herein, all the ligands are same. In some other embodiments of any one of the aspects described herein, the ligands are different.
  • At least one of R 7 and R 7’ is selected from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, polyethylene glycols (PEGs), and nitrogen protecting groups.
  • PEGs polyethylene glycols
  • R 7 and R 7’ is selected from the group consisting of targeting ligands, endosomolytic ligands and PK modulating ligands.
  • both of R 7 and R 7’ are not H.
  • R 7 and R 7’ can be same or different. In some embodiments of any one of the aspects, R 7 and R 7’ are same. In some embodiments of any one of the aspects, R 7 and R 7’ are different.
  • each R 7 and R 7’ is independently a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, C 1-30 alkyl, C 1-30 alkenyl, or C 1-30 alkynyl, each optionally substituted with one or more aldehyde (-C(O)H), carboxylic acid (-COOH), C 1-10 acyl (i.e., -C(O)C 1-10 alkyl), hydroxyl, halogen, cyano, nitro, azido, thiol (i.e, -SH), amino, C 1-10 alkoxy, C 1-10 alkylthio, C 1-10 alkylamino, di(C 1-10 alkyl)amino, C 1 - 10 alkylcarboxylate (i.e., -C(O)OC 1-10 alkyl), N-(C 1-10 alkyl)amide (i.e., a targeting ligand (e.g., GalNac),
  • At least one (e.g., both of) R 7 and R 7’ is selected independently from the group consisting of carbohydrates; peptides; lipids; diagnostic agents (biotin); fluorescent dyes; PEGs; antibody; antibody fragments (Fab, Nanobodies, etc); folic acid and vitamins; RGD-peptides; DUPA ligand, transferrin and transferrin receptor peptides, antibodies and their fragments; edosomolytic small molecules; and endosomolytic peptides.
  • one of R 7 and R 7’ is a targeting ligand and the other of R 7 and R 7’ is a pharmacokinetic modifier.
  • at least one of R 7 and R 7’ is a nitrogen protecting group.
  • R 7 and R 7’ together form a nitrogen protecting group.
  • At least one of R 7 and R 7’ is an antibody or an antigen binding fragment thereof.
  • at least one of R 7 and R 7’ is an antibody or an antigen binding fragment thereof and the other of R 7 and R 7’ is a lipid.
  • at least one of R 7 and R 7’ is an oligonucleotide.
  • R 7 and R 7’ is an antibody or an antigen binding fragment thereof and the other of R 7 and R 7’ is an oligonucleotide.
  • both of R 7 and R 7’ are indepently selected oligonucleotides.
  • L 2 can be a linker.
  • L 2 can be a bond.
  • L 2 is an optionally subtitued C 1 -C 20 alkylene, (e.g., –(CH 2 )b–, where b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 19 or 20), or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • L 2 is an optionally substituted subtitued C 1 - C6alkylene.
  • L 2 is an optionally subtitued C 1 - C20alkylene, where the backbone of the alkylene is interrupted with a heteroaryl (e.g., triazole) or NHC(O).
  • L 2 is optionally substituted C2alkylene, e.g., –CH 2 CH 2 –.
  • L 2 is a bond.
  • L 2 is –(CH 2 ) 9 –, –(CH 2 ) 10 –, –(CH 2 ) 11 –, –(CH 2 ) 12 –, or –(CH 2 ) 13 –.
  • L 2 is –(CH 2 )5–NHC(O)–(CH 2 )11–.
  • L2 is –CH 2 CH 2 O-(CH 2 )LM-CH 2 –, where LM is an intger selected from 1-15. For example, LM s 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • LM is an integer selected from 1 to 10.
  • LM is 1, 2, 3, 4, 5, 6, 7, 89 or 10.
  • L 2 is –CH 2 C(O)NH-(CH 2 ) LN -CH 2 –, where LN is an intger selected from 1-15.
  • LN is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • LN is an integer selected from 1 to 10.
  • LN is 1, 2, 3, 4, 5, 6, 7, 89 or 10.
  • L 2 comprises ring formed by an azide- alkyne cycloaddition reaction.
  • L 2 is an optionally substituted C 1 -C 20 alkylene, where the backbone of the alkylene is interrupted with a ring formed by an azide-alkyne cycloaddition reaction.
  • L 2 is ––(CH 2 )1-19–X–(CH 2 )1-19–, where X is a ring formed by an azide-alkyne cycloaddition reaction.
  • L 2 is ––CH 2 – X–(CH 2 ) 2 –, where X is a 1,2,3-triazole.
  • R 3NN is hydrogen, halogen, -OR 332 , -SR 333 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2- 30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O)rCH 2 CH 2 OR 334 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH)sCH 2 CH 2 -R 335 , NHC(O)R 336 , a lipid, a linker co
  • R 3NN is hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2- 30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O)rCH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH)sCH 2 CH 2 -R 325 , NHC(O)R 324 .
  • R 3NN is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2 - 30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl- ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ).
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido
  • R 3NN is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido
  • R 3NN is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, or alkoxyalkyl (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido).
  • R 3NN is hydrogen, hydroxyl, protected hydroxyl, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O-DMAEOE) or -O-N- methylacetamido (-O-NMA).
  • R 3NN is hydrogen, hydroxyl, protected hydroxyl or methoxy.
  • R 3NN is a reactive phosphorous group.
  • R 3NN is a solid support or a linker covalently attached to a solid support.
  • R 3 is –OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • only one of R 2 and R 3 is a solid support or a linker covalently attached to a solid support.
  • R 332 when R 3NN is –OR 332 , R 332 can be hydrogen or a hydroxyl protecting group.
  • R 332 can be hydrogen in some embodiments of any one of the aspects described herein.
  • R 3NN is – OC(O)CH 2 CH 2 CO 2 H.
  • R 333 can be hydrogen or a sulfur protecting group. Accordingly, in some embodiments of any one of the aspects, R 333 is hydrogen.
  • R 3NN is -O(CH 2 CH 2 O) r CH 2 CH 2 OR 334
  • r can be 1-50;
  • R 334 is independently for each occurrence H, C 1 -C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 335 ; and
  • R 335 is independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 3NN is -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 335
  • s can be 1-50 and R 335 can be independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 3NN is hydrogen, halogen, –OR 332 , or optionally substituted C 1 -C 30 alkoxy.
  • R 3 is halogen, –OR 332 , or optionally substituted C 1 -C 30 alkoxy.
  • R 3NN is F, OH or optionally substituted C 1 -C 30 alkoxy.
  • R 3NN is C 1 -C 30 alkoxy optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 3 is –O(CH 2 ) t CH 3 , where t is 1-21.
  • R 3NN is –O(CH 2 ) u R 337 , where u is 2- 10; R 337 is C 1 -C 6 alkoxy, amino (NH 2 ), CO 2 H, OH or halo.
  • R 337 is -CH 3 or NH 2 .
  • R 3NN is –O(CH 2 ) u - OMe or R 3NN is –O(CH 2 ) u NH 2 .
  • u is 2, 3, 4, 5 or 6.
  • u is 2, 3 or 6.
  • u is 2.
  • u is 3 or 6.
  • R 3NN is a C 1 - C 6 haloalkyl.
  • R 3NN is a C 1 -C 4 haloalkyl.
  • R 3 is –CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 or -CF 2 (CF 3 ) 2 .
  • R 3NN is – OCH(CH 2 OR 338 )CH 2 OR 339 , where R 338 and R 339 independently are H, optionally substituted C 1 - C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 338 and R 339 independently are optionally substituted C 1 -C 30 alkyl.
  • R 3NN is – CH 2 C(O)NHR 3310 , where R 3310 is H, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 - C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 5NN is R 6 , -Z-L 2 -R 6 , R 551 , hydrogen, hydroxyl, optionally substituted C 1-6 alkyl-R 551 , optionally substituted -C 2-6 alkenyl- R 551 , or optionally substituted -C 2-6 alkynyl-R 551 , where R 551 can be –OR 552 , -SR 553 , hydrogen, a phosphorous group, a solid support or a linker to a solid support. When R 551 is –OR 552 , R 552 can be H or a hydroxyl protecting group.
  • R 551 when R 551 is –SR 553 , R 553 can be H or a sulfur protecting group.
  • R 5NN is –OR 552 or - SR 553 .
  • R 5NN is –OR 552 and R 552 is 4,4′-dimethoxytrityl (DMT), e.g., R 5NN is –O-DMT.
  • DMT 4,4′-dimethoxytrityl
  • R 5NN is –CH(R 554 )- R 551 , where R 554 is hydrogen, halogen, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl, optionally substituted C 2 -C 30 alkynyl, or optionally substituted C 1 -C 30 alkoxy.
  • R 5NN is –CH(R 554 )-R 551
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 551 is a reactive phosphorous group.
  • At least one at least one R 555 in P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ) is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2 - 30 alkynyl, or an oxygen-protecting group.
  • At least one R 555 is H and at least one R 555 is other than H in -P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , and -SP(S)(SR 556 )(OR 555 ).
  • all R 555 are H in -P(O)(OR 555 ) 2 , - P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), - OP(S)(SR 556 ) 2 , -SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 555 are other than H in in - P(O)(OR 555 ) 2 , -P(S)(OR 555 ) 2 , -P(S)(SR 556 )(OR 555 ), -OP(O)(OR 555 ) 2 , -OP(S)(OR 555 ) 2 , - OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(O)(OR 555 ) 2 , -SP(S)(OR 555 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is H.
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is other than H.
  • At least one R 556 in - P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , - SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, or optionally substituted C 2-30 alkynyl, or an sulfur-protecting group.
  • At least one R 556 is H and at least one R 556 is other than H in -P(S)(SR 556 ) 2 , -OP(S)(SR 556 ) 2 and -SP(S)(SR 556 ) 2 .
  • all R 556 are H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , - OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • all R 556 are other than H in -P(S)(SR 556 )(OR 555 ), -P(S)(SR 556 ) 2 , -OP(S)(OR 555 ) 2 , -OP(S)(SR 556 )(OR 555 ), -OP(S)(SR 556 ) 2 , -SP(S)(SR 556 )(OR 555 ), and -SP(S)(SR 556 ) 2 .
  • R 3NN is a reactive phosphorous group, a solid support, a linker to a solid support, and R 5NN is a protected hydroxyl.
  • L 3 can be a linker.
  • L 3 can be a bond.
  • L 3 is an optionally subtitued C 1 -C 20 alkylene, (e.g., –(CH 2 )b–, where b is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 19 or 20), or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted
  • L 3 is an optionally substituted subtitued C 8 -C 16 alkylene, where the backbone of the alkylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), or C(O)O.
  • L 3 is –C(O)- (CH 2 )10–, –C(O)-(CH 2 )11–, or –C(O)-(CH 2 )5NHC(O)-(CH 2 )11–.
  • R AO [0299] In the various aspects described herein, each R AO is independently –O-L 3 -R 6 .
  • R AO is –O-L 3 -R 6 .
  • R AO is –O-L 3 -R 6 , where L 3 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 - C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R AO is –O-L 3 -R 6 , where L 3 is a polyethylene glycol (PEG).
  • L 3 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene
  • the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic
  • R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R AO is –O-L 3 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, polyethylene glycols (PEGs), nitrogen protecting group.
  • a ligand e.g., a ligand selected independently from the group consisting of carbohydrates,
  • R AO is –O-L 3 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 32 is hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino-O-C 4- 30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally
  • R 32 is a bond to an internucleotide linkage to a subsequent nucleotide, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • a 3’-oligonuclotide capping group e.g., an inverted nucleotide or an inverted abasic nucleotide
  • a solid support e.g., a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 32 is a bond to an internucleotide linkage to a subsequent nucleotide, a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 32 is a bond to an internucleotide linkage to a subsequent nucleotide. It is noted that only one of R 32 and R 33 can be a bond to an internucleotide linkage to a subsequent nucleotide.
  • R 32 is a solid support or a linker covalently attached to a solid support.
  • R 32 is –OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • R 32 is –OC(O)CH 2 CH 2 CO 2 H. It is noted that only one of R 32 and R 33 can be a solid support or a linker covalently attached to a solid support.
  • R 32 is –Z-L 2 -R 6 , hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH)sCH 2 CH 2 -R 325 , NHC(O)R 324 .
  • R 32 is–Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ).
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxy
  • R 32 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, alkoxyalkyl (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.
  • alkoxyalkyl e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido
  • alkoxyalkylamine e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmethoxyamido
  • alkoxyalkylamine e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N- methylmeth
  • R 32 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1-30 alkoxy, or alkoxyalkyl (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido).
  • R 32 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O- DMAEOE) or -O-N-methylacetamido (-O-NMA).
  • R 32 is –Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O- DMAEOE) or -O-N-methylacetamido (-O-NMA).
  • R 32 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O-DMAEOE) or - O-N-methylacetamido (-O-NMA).
  • R 32 is –Z-L 2 -R 6 .
  • R 32 is –Z-L 2 -R 6 , where Z is O.
  • R 32 is –Z-L 2 -R 6 .
  • R 32 is –Z-L 2 -R 6 , where L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 - C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R 32 is –Z-L 2 -R 6 , where L 2 is a polyethylene glycol (PEG).
  • R 32 is –Z-L 2 -R 6 , where Z is O and L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substitute
  • R 32 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ .
  • R 32 is –Z-L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1 - 30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl
  • a ligand e.g., a
  • R 32 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 32 and R 4 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y-C(R 10 R 11 ) v -2’; v is 1, 2 or 3; where Y is -O-, -CH 2 -, - CH(Me)-, -C(CH 3 ) 2 -, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, - N(R 12 )C(O)-, or -C(O)N(R 12 )-; R 10 and R 11 independently are H, optionally substituted C 1 -C 6 alkyl, optionally substituted C2-C6alkenyl or optionally substituted C2-C6alkynyl; R
  • R 32 and R 4 taken together are 4’- C(R 10 R 11 ) v -O-2’.
  • R 32 and R 4 taken together are 4’-CH 2 - O-2’, 4’-CH(CH 3 )-O-2’, 4’-CH(CH 2 OCH 3 )-O-2’, or 4’- CH 2 CH 2 -O-2’.
  • R 32 and R 4 taken together are 4’- CH 2 CH 2 -O-2’.
  • R 322 can be hydrogen or a hydroxyl protecting group.
  • R 323 can be hydrogen or a sulfur protecting group. Accordingly, in some embodiments of any one of the aspects, R 323 is hydrogen.
  • r can be 1-50; R 324 is independently for each occurrence H, C 1 -C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 325 ; and R 325 is independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 32 is -NH(CH 2 CH 2 NH) s CH 2 CH 2 -R 325 , s can be 1-50 and R 325 can be independently for each occurrence amino (NH 2 ), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.
  • R 32 is hydrogen, halogen, –OR 322 , or optionally substituted C 1 -C 30 alkoxy.
  • R 32 is halogen, –OR 322 , or optionally substituted C 1 -C 30 alkoxy.
  • R 32 is F, OH or optionally substituted C 1 -C 30 alkoxy.
  • R 32 is C 1 -C 30 alkoxy optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 2 is –O(CH 2 ) t CH 3 , where t is 1-21.
  • t is 14, 15, 16, 17 or 18.
  • t is 16.
  • R 32 is –O(CH 2 ) u R 327 , where u is 2-10; R 327 is C 1 -C 6 alkoxy, amino (NH 2 ), CO 2 H, OH or halo.
  • R 327 is -CH 3 or NH 2 .
  • R 32 is –O(CH 2 ) u - OMe or R 32 is –O(CH 2 ) u NH 2.
  • R 32 is a C 1 - C 6 haloalkyl.
  • R 32 is a C 1 -C 4 haloalkyl.
  • R 32 is –CF 3 , -CF 2 CF 3 , -CF 2 CF 2 CF 3 or -CF 2 (CF 3 ) 2 .
  • R 32 is – OCH(CH 2 OR 328 )CH 2 OR 329 , where R 328 and R 329 independently are H, optionally substituted C 1 - C 30 alkyl, optionally substituted C 2 -C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 328 and R 329 independently are optionally substituted C 1 -C 30 alkyl.
  • R 32 is – CH 2 C(O)NHR 3210 , where R 3210 is H, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 - C 30 alkenyl or optionally substituted C 2 -C 30 alkynyl.
  • R 3210 is H or optionally substituted C 1 -C 30 alkyl.
  • R 3210 is optionally substituted C 1 -C 6 alkyl.
  • R 33 is hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino-O-C 4- 30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally
  • R 33 is a bond to an internucleotide linkage to a subsequent nucleotide, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • a 3’-oligonuclotide capping group e.g., an inverted nucleotide or an inverted abasic nucleotide
  • a solid support e.g., a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 33 is a bond to an internucleotide linkage to a subsequent nucleotide, a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 33 is a bond to an internucleotide linkage to a subsequent nucleotide.
  • R 33 is a solid support or a linker covalently attached to a solid support.
  • R 33 is –OC(O)CH 2 CH 2 C(O)NH-Z, where Z is a solid support.
  • R 33 is –OC(O)CH 2 CH 2 CO 2 H.
  • R 33 is –Z-L 2 -R 6 , hydrogen, halogen, -OR 322 , -SR 323 , optionally substituted C 1-30 alkyl, C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or optionally substituted C 1-30 alkoxy, amino (NH 2 ), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH 2 CH 2 O) r CH 2 CH 2 OR 324 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH 2 CH 2 NH)s
  • R 33 is–Z-L 2 -R 6 , hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl- ON(CH 2 R 8 )(CH 2 R 9 ), or -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ).
  • C 1-30 alkoxy e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido
  • alkoxyalkyl alkoxyalkylamine
  • alkoxycarboxylate amino, alkylamino, dialkylamino,
  • R 33 is hydrogen, hydroxyl, protected hydroxyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.
  • C 1-30 alkoxy e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido
  • alkoxyalkyl alkoxyalkylamine
  • alkoxycarboxylate amino, alkylamino, or dialkylamino.
  • R 33 is –Z-L 2 -R 6 , hydroxyl, protected hydroxyl, or optionally substituted C 1-30 alkoxy, (e.g., methoxy, 2-methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido).
  • R 33 is –Z-L 2 -R 6 , hydroxyl, protected hydroxyl, methoxy, 2-methoxyethoxy, -O-dimethylaminoethoxyethyl (-O-DMAEOE) or -O-N- methylacetamido (-O-NMA).
  • R 33 is –Z-L 2 -R 6 .
  • R 33 is –Z-L 2 -R 6 , where Z is O.
  • R 33 is –Z-L 2 -R 6 .
  • R 33 is –Z-L 2 -R 6 , where L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 - C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R 33 is –Z-L 2 -R 6 , where L 2 is a polyethylene glycol (PEG).
  • R 33 is –Z-L 2 -R 6 , where Z is O and L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substitute
  • R 33 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ .
  • R 33 is –Z-L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1 - 30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl
  • a ligand e.g., a
  • R 33 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 33 and R 4 taken together are 4’-C(R 10 R 11 ) v -Y-2’ or 4’-Y-C(R 10 R 11 ) v -2’; v is 1, 2 or 3; where Y is -O-, -CH 2 -, - CH(Me)-, -C(CH 3 ) 2 -, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, - N(R 12 )C(O)-, or -C(O)N(R 12 )-; R 10 and R 11 independently are H, optionally substituted C 1 -C 6 alkyl, optionally substituted C 2 -C 6 alkenyl or optionally substituted C 2 -C 6 alkyn
  • R 32 and R 4 taken together are 4’- C(R 10 R 11 ) v -O-2’.
  • R 32 and R 4 taken together are 4’-CH 2 - O-2’, 4’-CH(CH 3 )-O-2’, 4’-CH(CH 2 OCH 3 )-O-2’, or 4’- CH 2 CH 2 -O-2’.
  • R 32 and R 4 taken together are 4’- CH 2 CH 2 -O-2’.
  • R 33M is hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino-O-C 4- 30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ),
  • R 33M is a bond to an internucleotide linkage to a subsequent nucleotide, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • a 3’-oligonuclotide capping group e.g., an inverted nucleotide or an inverted abasic nucleotide
  • a solid support e.g., a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 33M is a bond to an internucleotide linkage to a subsequent nucleotide, a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 33M is a bond to an internucleotide linkage to a subsequent nucleotide.
  • R 33M is a solid support or a linker covalently attached to a solid support.
  • R 33M is –OC(O)CH 2 CH 2 C(O)NH- Z, where Z is a solid support. In some embodiments, R 33M is –OC(O)CH 2 CH 2 CO 2 H. [0361] In some embodiments of any one of the aspects described herein, R 33M is –Z-R 33L . For example, R 33M is –O-R 33L . [0362] In some embodiments of any one of the aspects described herein, R 33L is a ligand or a linker covalently linked to one or more ligands.
  • R 33L is optionally substituted C 1-30 alkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, or polyethylene glycol.
  • R 33L is alkyl, alkyl ester, mono-GalNac, Tri-GalNac, hetero-alky, alkyne, alkene, alkylether, PEG, Biotin, Vit-E, alkyl folate, cyclic RGD.
  • R 333L is a peptide.
  • R 35 is R 6 , -Z-L 2 -R 6 , a bond to an internucleotide linkage to a preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, phosphate group, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl- ON(CH 2 R 8 )(CH 2 R 9 ), vinylphosphonate (VP) group, C
  • R 35 is a bond to an internucleotide linkage to a preceding nucleotide.
  • R 35 is R 6 .
  • R 35 is -O-N(R 7 )R 7’ .
  • R 35 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, polyethylene glycols (PEGs), nitrogen protecting group.
  • a ligand e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins,
  • R 35 is -O-N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 35 is –Z-L 2 -R 6 .
  • R 5 is – Z-L 2 -R 6 , where Z is O.
  • R 35 is –Z-L 2 -R 6 .
  • R 5 is – Z-L 2 -R 6 , where L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substituted aliphatic.
  • R 35 is –Z-L 2 -R 6 , where L 2 is a polyethylene glycol (PEG).
  • R 35 is –Z-L 2 -R 6 , where Z is O and L 2 is optionally substituted C 1 -C 20 alkylene, optionally substituted C 2 -C 20 alkenylene or optionally substituted C 2 -C 20 alkynylene, and where the backbone of the alkylene, alkenylene or alkynylene can be interrupted or terminated by O, S, S(O), SO 2 , NR 1 , NR 1 -C(O), C(O), C(O)O, cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R N1 is hydrogen, acyl, aliphatic or substitute
  • R 35 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ .
  • R 35 is –Z-L 2 -R 6 , where R 6 is -O-N(R 7 )R 7’ , where R 7 and R 7’ are independently H or a ligand, (e.g., a ligand selected independently from the group consisting of carbohydrates, lipids, vitamins, peptides, proteins, lipoproteins, peptidomimetics, polyamines, nucleosides and nucleotides, oligonucleotides, therapeutic agents, diagnostic agents, detectable labels, antibodies or fragments thereof, optionally substituted C 1-30 alkyl, optionally substituted C 1- 30 alkenyl, optionally substituted C 1-30 alkynyl, optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl, optional
  • R 35 is –Z-L 2 -R 6 , where R 6 is -O- N(R 7 )R 7’ , where R 7 and R 7’ together with the N they are attached to form an optionally substituted heterocyclyl (e.g., phthalimide or morpholine).
  • R 35 is –OR 552 or - SR 553 .
  • R 35 is –OR 552 and R 552 is 4,4′-dimethoxytrityl (DMT), e.g., R 35 is –O-DMT.
  • R 35 is –CH(R 554 )- R 551 , where R 554 is hydrogen, halogen, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl, optionally substituted C 2 -C 30 alkynyl, or optionally substituted C 1 -C 30 alkoxy.
  • R 35 is –CH(R 554 )-R 551
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 43N is hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, a reactive phosphorous group, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy, dimethylaminoethoxyethyoxy, N-methylmethoxyamido), alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino-O-C 4- 30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ), -O-C 4-30 alkyl-ON(CH 2 R 8 )(CH 2 R 9 ),
  • R 43N is a bond to an internucleotide linkage to a subsequent nucleotide, a 3’-oligonuclotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a solid support, or a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • a 3’-oligonuclotide capping group e.g., an inverted nucleotide or an inverted abasic nucleotide
  • a solid support e.g., a linker covalently bonded (e.g., -C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 43N is a bond to an internucleotide linkage to a subsequent nucleotide, a solid support, or a linker covalently bonded (e.g., - C(O)CH 2 CH 2 C(O)-) to a solid support.
  • R 43N is a bond to an internucleotide linkage to a subsequent nucleotide.
  • R 43N is a solid support or a linker covalently attached to a solid support.
  • R 43N is –OC(O)CH 2 CH 2 C(O)NH- Z, where Z is a solid support. In some embodiments, R 43N is –OC(O)CH 2 CH 2 CO 2 H.
  • R 45N is a bond to an internucleotide linkage to a preceding nucleotide, hydrogen, hydroxyl, protected hydroxyl, phosphate group, optionally substituted C 1-30 alkyl, optionally substituted C 1-30 haloalkyl, optionally substituted C 2-30 alkenyl, optionally substituted C 2-30 alkynyl, optionally substituted C 1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C 4-30 alkyl-ON(CH 2 R 8 )(
  • R 45N is a bond to an internucleotide linkage to a preceding nucleotide.
  • R 45N is –OR 552 or - SR 553 .
  • R 45N is –OR 552 and R 552 is 4,4′-dimethoxytrityl (DMT), e.g., R 45N is –O-DMT.
  • DMT 4,4′-dimethoxytrityl
  • R 45N is –CH(R 554 )- R 551 , where R 554 is hydrogen, halogen, optionally substituted C 1 -C 30 alkyl, optionally substituted C 2 -C 30 alkenyl, optionally substituted C 2 -C 30 alkynyl, or optionally substituted C 1 -C 30 alkoxy.
  • R 45N is –CH(R 554 )-R 551
  • R 554 is H.
  • R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • R 554 is H. In some other non-limiting examples, R 554 is C 1 -C 30 alkyl optionally substituted with a NH 2 , OH, C(O)NH 2 , COOH, halo, SH, or C 1 -C 6 alkoxy.
  • J in compouds of Formulae Ig, Ii, IIg and IVg, J can be O, S, CH 2 or N-alkyl (e.g., NCH3).
  • J In some embodiments of any one of the aspects, J is O.
  • J In some embodiments of any one of the aspects, J is S.
  • J In some embodiments of any one of the aspects, J is CH 2 .
  • Internucleoside linkages refers to a covalent linkage between adjacent nucleosides.
  • the two main classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom.
  • Representative phosphorus containing linkages include, but are not limited to, phosphodiesters (P ⁇ O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P ⁇ S).
  • Non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)— S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); and N,N′- dimethylhydrazine (—CH2-N(CH3)-N(CH3)-).
  • Modified internucleoside linkages compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide compound.
  • linkages having a chiral atom can be prepared as racemic mixtures, as separate enantiomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous- containing and non-phosphorous-containing linkages are well known to those skilled in the art.
  • the phosphate group in the internucleoside linkage can be modified by replacing one of the oxygens with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown.
  • modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • one of the non-bridging phosphate oxygen atoms in the phosphodiester internucleoside linkage can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an aryl group, etc...), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl).
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral. In other words a phosphorous atom in a phosphate group modified in this way is a stereogenic center.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).
  • Phosphorodithioates have both non-bridging oxygens replaced by sulfur.
  • the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers.
  • non-bridging oxygens which eliminate the chiral center, e.g. phosphorodithioate formation
  • the non-bridging oxygens can be independently any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).
  • R is alkyl or aryl.
  • a phosphodiester internucleoside linkage can also be modified by replacement of bridging oxygen, (i.e. oxygen that links the phosphate to the sugar of the nucleosides), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • the replacement can occur at the either one of the linking oxygens or at both linking oxygens.
  • the bridging oxygen is the 3’-oxygen of a nucleoside, replacement with carbon is preferred.
  • the bridging oxygen is the 5’-oxygen of a nucleoside, replacement with nitrogen is preferred.
  • Modified phosphate linkages where at least one of the oxygen linked to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorous group, are also referred to as “non-phosphodiester intersugar linkage” or “non-phosphodiester linker.”
  • the phosphate group can be replaced by non-phosphorus containing connectors, e.g. dephospho linkers.
  • Dephospho linkers are also referred to as non- phosphodiester linkers herein. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.
  • Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amides, carbamate and ethylene oxide linker.
  • a modification of a non-bridging oxygen can necessitate modification of 2’-OH, e.g., a modification that does not participate in cleavage of the neighboring intersugar linkage, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA.
  • Preferred non-phosphodiester internucleoside linkages include phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more enantiomeric excess of Sp isomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more enantiomeric excess of Rp isomer, phosphorodithioates, phsophotriesters, aminoalkylphosphotrioesters, alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidate), and boranophosphonates.
  • the oligonucleotides described herein comprise one or more neutral internucleoside linkages that are non-ionic.
  • non-phosphodiester backbone linkage is selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate and phosphoramidate backbone linkages.
  • the internucleoside linkage is where R IL1 and R IL2 are each independently for each occurrence absent, O, S, CH 2 , NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, wherein backbone of the alkylene can comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and/or at the end; and R IL3 and R IL4 are each independently selected from the group consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR3 (R is hydrogen, alkyl, aryl), BH 3 - , C (i.e.
  • R IL1 and R IL2 are O.
  • R IL1 and R IL2 are O and at least one of R IL3 and R IL4 is other than O.
  • one of R IL3 and R IL4 is S and the other is O or both of R IL3 and R IL4 are S.
  • one of R 33 or R 35 is a bond to a modified internucleoside linkage, e.g., an internucleoside linkage of structure: , where at least one of R IL1 , R IL2 , R IL3 and R IL4 is not O.
  • at least one of R IL3 and R IL4 is S.
  • both of R 33 and R 35 are a bond to a modified internucleoside linkage.
  • R 33 is a bond to phosphodiester internucleoside linkage.
  • R 35 is a bond to phosphodiester internucleoside linkage.
  • R 33 is a bond to a modified internucleoside linkage and R 35 is a bond to phosphodiester internucleoside linkage.
  • R 35 is a bond to a modified internucleoside linkage and R 33 is a bond to phosphodiester internucleoside linkage.
  • one of R 33M or R 35 is a bond to a modified internucleoside linkage, e.g., an internucleoside linkage of structure: , where at least one of R IL1 , R IL2 , R IL3 and R IL4 is not O. For example, at least one of R IL3 and R IL4 is S.
  • both of R 33M and R 35 are a bond to a modified internucleoside linkage.
  • R 33M is a bond to phosphodiester internucleoside linkage.
  • R 35 is a bond to phosphodiester internucleoside linkage.
  • R 33M is a bond to a modified internucleoside linkage and R 35 is a bond to phosphodiester internucleoside linkage.
  • R 35 is a bond to a modified internucleoside linkage and R 33M is a bond to phosphodiester internucleoside linkage.
  • one of R 43N or R 45N is a bond to a modified internucleoside linkage, e.g., an internucleoside linkage of structure: , where at least one of R IL1 , R IL2 , R IL3 and R IL4 is not O. For example, at least one of R IL3 and R IL4 is S.
  • both of R 43N and R 45N are a bond to a modified internucleoside linkage.
  • R 43N is a bond to phosphodiester internucleoside linkage.
  • R 45N is a bond to phosphodiester internucleoside linkage.
  • R 43N is a bond to a modified internucleoside linkage and R 45N is a bond to phosphodiester internucleoside linkage.
  • R 45N is a bond to a modified internucleoside linkage and R 43N is a bond to phosphodiester internucleoside linkage.
  • the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more modified internucleoside linkages.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5 or 6 modified internucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3 or 4 modified internucleoside linkages.
  • the oligonucleotide comprises at least two modified internucleoside linkages between the first five nucleotides counting from the 5’-end of the oligonucleotide and further comprises at least two modified internucleoside linkages between the first five nucleotides counting from the 3’-end of the oligonucleotide.
  • the oligonucleotide comprises modified internucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3’-end of the oligonucleotide.
  • the modified internucleoside linkage is a phosphorothioate.
  • the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3, 4, 5 or 6 phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises 1, 2, 3 or 4 phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 5’-end of the oligonucleotide and further comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 3’-end of the oligonucleotide.
  • the oligonucleotide comprises modified internucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3’-end of the oligonucleotide.
  • Oxygen protecting groups [0445] Some embodiments of the various aspects described herein include an oxygen protecting group (also referred to as an hydroxyl protecting group herein).
  • Oxygen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • oxygen protecting groups include, but are not limited to, methyl, t- butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM), methylthiomethyl (MTM), t- butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexy
  • oxygen protecting group is benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4′-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p- methoxyphenyl)xanthine-9-yl (MOX).
  • DMT 4,4′-dimethoxytrityl
  • Pixyl 9-phenylxanthine-9-yl
  • MOX 9-(p- methoxyphenyl)xanthine-9-yl
  • the hydroxyl protecting group is selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4′-dimethoxytrityl.
  • protected hydroxy and “protected hydroxyl” as used herein mean a group of the formula -OR Pro , wherein R Pro is an oxygen protecting group as defined herein.
  • Nitrogen protecting groups [0450] Some embodiments of the various aspects described herein include a nitrogen protecting group (also referred to as an amino protecting group herein).
  • Nitrogen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5 th Edition, John Wiley & Sons, 2014, incorporated herein by reference.
  • Additional exemplary nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacyl derivative, N′- phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N- 2,3- diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5- triazacyclohexan-2-one, 5-substituted 1,3- dibenzyl-1,3,5-triazacyclohexan-2-one, 1- substituted 3,5
  • nucleoside Formula IIIa-IIIg or IVa-IVg can be located anywhere in the oligonucleotide. In some embodiments, the nucleoside of Formula IIIa-IIIg or IVa-IVg is present at the 5’- or 3’-terminus of the oligonucleotide.
  • the nucleoside of Formula IIIa-IIIg or IVa-IVg is present at an internal position of the oliogunculeotide.
  • the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula IIIa-IIIg or IVa-IVg, a nucleoside with a modified sugar.
  • a “modified sugar” is meant a sugar or moiety other than 2’-deoxy (i.e, 2’-H) or 2’-OH ribose sugar.
  • nucleotides comprising a modified sugar are 2’-F ribose, 2’-OMe ribose, 2’-O,4’-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA), cyclohexene (Cyclohexene nucleic acid, CeNA), 2’- methoxyethyl ribose, 2’-O-allyl ribose, 2’-C-allyl ribose, 2'-O-N-methylacetamido (2'-O-NMA) ribose, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) ribose, 2'-O-aminopropyl (2'-O-AP) ribose, 2’-F arabinose (2'-ara-F
  • the nucleoside with the modified sugar can be present at any position of the oligonucleotide.
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro (2’-F) nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 102’-F nucleotides.
  • the 2’-F nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formulae IIIa-IIIg and IVa-IVg and 2’-F nucleosides.
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2’-OMe nucleotides. It is noted that the 2’-OMe nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formulae IIIa-IIIg and IVa-IVg and 2’-OMe nucleosides. In some other embodiments, the oligonucleotide comprises, e.g., solely comprises solely comprises nucleosides of Formulae IIIa-IIIg and IVa-IVg, 2’-OMe nucleosides and 2’-F nucleosides.
  • the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-deoxy, e.g., 2’-H nucleotides.
  • the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of 2’-deoxy, e.g., 2’-H nucleotides. It is noted that the 2’- deoxy, e.g., 2’-H nucleotides can be present at any position of the oligonucleotide.
  • the oligonucleotide can comprise a 2’-deoxy, e.g., 2’-H nucleotide at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the oligonucleotide.
  • the oligonucleotide comprises a 2’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the oligonucleotide.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formulae IIIa-IIIg and IVa-IVg and 2’-deoxy (2’-H) nucleotides.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula IIIa- IIIg or IVa-IVg, 2’-OMe nucleosides, and 2’-deoxy (2’-H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formulae IIIa-IIIg and IVa-IVg, 2’-F nucleosides and 2’-deoxy (2’-H) nucleotides.
  • the oligonucleotide comprises, e.g., solely comprises nucleosides of Formulae IIIa-IIIg and IVa-IVg, 2’-OMe nucleosides, 2’-F nucleosides and 2’-deoxy (2’-H) nucleotides.
  • the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula IIIa-IIIg or IVa-IVg, a non-natural nucleobase.
  • the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising an independently selected non-natural nucleobase.
  • a nucleotide comprising a non-natural nucleobase can be present anywhere in the oligonucleotide.
  • the oligonucleotide further comprises a solid support linked thereto.
  • the oligonucleotides described herein can range from few nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) in length to hunderes of nucleotides in length.
  • the oligonucleotide can be from 5 nucleotides to 100 nucleotides in length. In some embodiments, the oligonucleotide is from 10 nucleotides to 50 nucleotides in length. For example, the oligonucleotide is between 15 and 35, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. In some embodiments, longer oligonucleotides of between 25 and 30 nucleotides in length are preferred. In some embodiments, shorter oligonucleotides of between 10 and 15 nucleotides in length are preferred. In another embodiment, the oligonucleotide is at least 21 nucleotides in length.
  • the oligonucleotide described herein comprises a pattern of backbone chiral centers.
  • a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration, and no more than 8 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration, and no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration, and no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration, and no more than 4 intemucleotidic linkages which are not chiral.
  • the intemucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous.
  • the intemucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous.
  • the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.
  • the oligonucleotide described herein comprises a stereochemistry block.
  • a block is an Rp block in that each internucleotidic linkage of the block is Rp.
  • a 5’-block is an Rp block.
  • a 3’-block is an Rp block.
  • a block is an Sp block in that each internucleotidic linkage of the block is Sp.
  • a 5’-block is an Sp block.
  • a 3’-block is an Sp block.
  • provided oligonucleotides comprise both Rp and Sp blocks.
  • provided oligonucleotides comprise one or more Rp but no Sp blocks.
  • provided oligonucleotides comprise one or more Sp but no Rp blocks.
  • provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.
  • the oligonculeotide described herein comprises a 5’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification.
  • a 5’-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’- block is an Sp block wherein each of internucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’-block comprises 4 or more nucleoside units.
  • a 5’-block comprises 5 or more nucleoside units. In some embodiments, a 5’-block comprises 6 or more nucleoside units. In some embodiments, a 5’-block comprises 7 or more nucleoside units.
  • a 3’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3’-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3’-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 3’-block comprises 4 or more nucleoside units.
  • a 3’-block comprises 5 or more nucleoside units.
  • a 3’-block comprises 6 or more nucleoside units.
  • a 3’-block comprises 7 or more nucleoside units.
  • oligonucleotide described herein comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc.
  • A is followed by Sp.
  • A is followed by Rp.
  • A is followed by natural phosphate linkage (PO).
  • U is followed by Sp.
  • U is followed by Rp.
  • U is followed by natural phosphate linkage (PO).
  • C is followed by Sp.
  • C is followed by Rp.
  • C is followed by natural phosphate linkage (PO).
  • G is followed by Sp.
  • G is followed by Rp.
  • G is followed by natural phosphate linkage (PO).
  • C and U are followed by Sp.
  • C and U are followed by Rp.
  • C and U are followed by natural phosphate linkage (PO).
  • a and G are followed by Sp.
  • a and G are followed by Rp.
  • the oligonucleotides described herein are 5’ phosphorylated or include a phosphoryl analog at the 5’ prime terminus.
  • 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5'-monophosphate ((HO) 2 (O)P-O-5'); 5'-diphosphate ((HO) 2 (O)P- O-P(HO)(O)-O-5'); 5'-triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'- adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'- (HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)P- O-5'); 5
  • exemplary 5’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO) 2 (X)P-O[-(CH 2 )a-O-P(X)(OH)-O]b- 5', ((HO)2(X)P-O[-(CH 2 )a- P(X)(OH)-O]b- 5', ((HO)2(X)P-[-(CH 2 )a-O-P(X)(OH)-O]b- 5'; dialkyl terminal phosphates and phosphate mimics:
  • the oligonucleotide comprises a 5’-vinylphosphonate group.
  • the oligonucleotide comprises a 5’-E-vinyl phosphonate group.
  • the oligonucleotide comprises a 5’-Z- vinylphosphonate group.
  • the oligonucleotide dscribed herein comprises a 5’-morpholino, a 5’-dimethylamino, a 5’-deoxy, an inverted abasic, or an inverted abasic locked nucleic acid modification at the 5’-end.
  • the oligonucleotide dscribed herein can comprise a thermally destabilizing modification.
  • the oligonucleotide can comprise at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5’-end of the oligonucleotide.
  • the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5’-end of the antisense strand. In some embodiments, thermally destabilizing modification is located in positions 2-9, or preferably positions 4-8, counting from the 5’-end of the oligonucleotide. In some further embodiments, the thermally destabilizing modification is located at position 5, 6, 7 or 8, counting from the 5’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 7, counting from the 5’-end of the oligonucleotide.
  • thermally destabilizing modification(s) includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s).
  • Tm overall melting temperature
  • the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5’-end of the antisense strand.
  • the thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).
  • UUA unlocked nucleic acids
  • GNA glycol nucleic acid
  • the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA.
  • the destabilizing modification mUNA is selected from the group consisting of
  • R H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-nPr; O- alkyl; O-alkylamino;
  • R' H, Me;
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C
  • R H, OH; OMe; Cl, F; OH; O-(CH 2 ) 2 OMe; SMe, NMe 2 ; NH 2 ; Me; CCH (alkyne), O-nPr; O- alkyl; O-alkylamino;
  • R' H, Me;
  • B A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2- modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C
  • Exemplified sugar modifications include, but are not limited to the following: wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.
  • the thermally destabilizing modification of the duplex is selected from the mUNA and GNA building blocks described in Examples 1-3 herein.
  • the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5’-mUNA, 4’-mUNA, 3’-mUNA, and 2’-mUNA.
  • the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GNA, 2’-OMe, 3’-OMe, 5’-Me, Hy p-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h’GNA (Mod A-Mod K).
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are independently or in combination absent from the nucleotide.
  • bonds between the ribose carbons e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’
  • bonds between the ribose carbons e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4
  • acyclic nucleotide is or wherein B is a modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • the term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between C1'-C4' being removed (i.e. the covalent carbon- oxygen-carbon bond between the C1' and C4' carbons).
  • the C2'-C3' bond i.e. the covalent carbon-carbon bond between the C2' and C3' carbons
  • the acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings.
  • the acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.
  • the term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds: .
  • the thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex.
  • mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof.
  • Other mismatch base pairings known in the art are also amenable to the present invention.
  • a mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides.
  • the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand.
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as: .
  • More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.
  • the thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.
  • the thermally destabilizing modification includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more ⁇ -nucleotide complementary to the base on the target mRNA, such as: wherein R is H, OH, OCH 3 , F, NH 2 , NHMe, NMe 2 or O-alkyl
  • R is H, OH, OCH 3 , F, NH 2 , NHMe, NMe 2 or O-alkyl
  • Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are: [0499]
  • the alkyl for the R group can be a C 1 -C 6 alkyl.
  • the oligonucleotide can comprise one or more stabilizing modifications.
  • the oligonucleotide can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • the oligonucleotide comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • a stabilizing modification in the oligonucleotide can be present at any positions.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 6, 14 and 16, counting from the 5’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 2, 14 and 16, counting from the 5’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 7, 10 and 11, counting from the 5’-end.
  • the oligonucleotide comprises stabilizing modifications at positions 7, 9, 10 and 11, counting from the 5’-end.
  • the oligonucleotide comprises at least one stabilizing modification adjacent to a destabilizing modification.
  • the stabilizing modification can be the nucleotide at the 5’-end or the 3’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification.
  • the oligonucleotide comprises a stabilizing modification at each of the 5’-end and the 3’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the oligonucleotide comprises at least two stabilizing modifications at the 3’-end of a destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • Exemplary thermally stabilizing modifications include, but are not limited to 2’-fluoro modifications.
  • Other thermally stabilizing modifications include, but are not limited to LNA.
  • Double-stranded RNAs [0505] The skilled person is well aware that double-stranded RNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888).
  • dsRNA double-stranded RNA
  • a first strand also referred to as an antisense strand or a guide strand
  • a second strand also referred to as a sense strand or passenger strand
  • at least one of the first (i.e., the antisense strand) or the second strand (i.e., the sense strand) is an oligonucleotide described herein.
  • the sense strand is an oligonucleotide described herein.
  • the sense strand comprises at least one nucleotide of Formula IIIa-IIIg or IVa-IVg.
  • the antisense strand is an oligonucleotide described herein.
  • the antisense strand comprises at least one nucleotide of Formula IIIa-IIIg or IVa-IVg.
  • the antisense strand is substantially complementary to a target nucleic acid, e.g., a target gene or mRNA gene and the dsRNA is capable of inducing targeted cleavage of the target nucleic acid.
  • a target nucleic acid e.g., a target gene or mRNA gene
  • the dsRNA is capable of inducing targeted cleavage of the target nucleic acid.
  • Each strand of the dsRNA molecule can range from 15-35 nucleotides in length.
  • each strand can be between, 17-35 nucleotides in length, 17-30 nucleotides in length, 25- 35 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19- 21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.
  • the sense and antisense strands can be equal length or unequal length.
  • the sense strand and the antisense strand independently have a length of 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
  • the antisense strand is of length 15-35 nucleotides.
  • the antisense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19- 25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length.
  • the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.
  • the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length.
  • the antisense strand is 22, 23 or 24 nucleotides in length.
  • the antisense strand is 23 nucleotides in length.
  • the sense strand can be, in some embodiments, 15-35 nucleotides in length.
  • the sense strand is 15-35, 17-35, 17-30, 25-35, 27- 30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length.
  • the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.
  • the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 19, 20, 21, 22 or 23 nucleotides in length.
  • the sense strand is 20, 21 or 22 nucleotides in length.
  • the sense strand is 21nucleotides in length [0513]
  • the sense strand can be 15-35 nucleotides in length
  • the antisense strand can be independent from the sense strand, 15-35 nucleotides in length.
  • the sense strand is 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length
  • the antisense strand is independently 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21- 25, 21-25, or 21-23 nucleotides in length.
  • the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.
  • the sense strand and the antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 19, 20, 21, 22 or 23 nucleotides in length and the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length.
  • the sense strand is 20, 21 or 22 nucleotides in length and the antisense strand is 22, 23 or 24 nucleotides in length.
  • the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.
  • the sense strand and antisense strand typically form a double-stranded or duplex region.
  • the duplex region of a dsRNA agent described herein can be 12-35 nucleotide (or base) pairs in length.
  • the duplex region can be between 14-35 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotides in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19- 21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length.
  • the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length. In some embodiments, the duplex region is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length. For example, the duplex region is 19, 20, 21, 22 or 23 nucleotide pairs in length. In some embodiments, the the duplex region is 20, 21 or 22 nucleotide pairs in length. For example, the dsRNA molecule has a duplex region of 21 base pairs.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of nucleotide of Formulae IIIa-IIIg and IVa-IVg.
  • the nucleotides of Formulae IIIa-IIIg and IVa-IVg all can be present in one strand.
  • the nucleotide of Formulae IIIa-IIIg and IVa-IVg may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides of Formula IIIa-IIIg or IVa-IVgdescribed herein.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can be present at any position of the sense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can be present at a terminal region of the sense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can be present at one or more of positions 1, 2, 3 and 4, counting from the 5’-end of the sense strand.
  • nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can be present at one or more of positions 1, 2, 3 and 4, counting from the 3’-end of the sense strand. In some embodiments, the nucleotide of Formula IIIa-IIIg or IVa-IVg can be present at one or more of positions 18, 19, 20 and 21, counting from 5’-end of the sense strand. The nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can also be located at a central region of sense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can be located at one or more of positions 6, 7, 8, 9, 10, 11, 12 and 13, counting from 5’-end of the sense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg is at the 5- terminus of the sense strand.
  • the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of nucleotides of Formula IIIa-IIIg or IVa-IVg described herein.
  • the nucleotide of Formula IIIa- IIIg or IVa-IVg described herein can be present at any position of the antisense strand.
  • nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can be present at a terminal region of the antisense strand.
  • nucleotide of Formula IIIa-IIIg or IVa-IVg described herein can be present at one or more of positions 1, 2, 3 and 4, counting from the 5’-end of the antisense strand.
  • nucleotide of Formula IIIa-IIIg or IVa-IVg described herein nucleotide can be present at one or more of positions 1, 2, 3, 4, 5 and 6, counting from the 3’-end of the antisense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg described herein nucleotide can be present at one or more of positions 18, 19, 20, 21, 22 and 23, counting from 5’-end of the antisense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg described herein nucleotide can also be located at a central region of the antisense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg described herein nucleotide can be located at one or more of positions 6, 7, 8, 9, 10, 11, 12 and 13, counting from 5’-end of the antisense strand.
  • the nucleotide of Formula IIIa-IIIg or IVa-IVg is at the 3’-termnus of the antisense strand.
  • the dsRNA agent can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising a modified sugar.
  • the dsRNA agent can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides independently selected from the group consisting of 2’-F, 2-OMe, acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2’-methoxyethyl, 2’-O-allyl, 2’-C-allyl, 2'-O-N- methylacetamido (2'-O-NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O- aminopropyl (2'-O-AP), and 2'-ara-F.
  • LNA locked nucleic acid
  • CeNA locked nucleic acid
  • CeNA 2’-methoxyethyl
  • 2’-O-allyl 2’-C-allyl
  • 2'-O-N-NMA 2'-O-dimethylaminoethoxyeth
  • a nucleotide comprising modified sugar can be present anywhere in the dsRNA molecule.
  • a nucleotide comprising a modified sugar can be present in the sense strand or a nucleotide comprising a modified sugar can be present in the antisense strand.
  • two or more nucleotides comprising a modified sugar are present in the dsRNA molecule, they can all be in the sense strand, antisense strand or both in the sense and antisense strands.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro (2’-F) nucleotides.
  • the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro nucleotides.
  • the 2’-fluoro nucleotides can be located anywhere in the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at position 10, counting from 5’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at position 10, counting from 5’- end of the sense strand and the sense strand further comprises a 2’-fluoro nucleotide at position 8, 9, 11 or 12, counting from 5’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at positions 9 10, counting from 5’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at positions 10 and 11, counting from 5’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at positions 9, 10 and 11, counting from 5’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at positions 8, 9 and 10, counting from 5’-end of the sense strand.
  • the sense strand comprises a 2’-fluoro nucleotide at positions 10, 11 and 12, counting from 5’-end of the sense strand.
  • the antisense comprises 2’-fluoro nucleotides at positions 7, 10 and 11 from the 5’-end.
  • the sense strand comprises 2’-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5’-end.
  • the sense strand comprises 2’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5’-end of the antisense strand.
  • the sense strand comprises 2’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four 2’-fluoro nucleotides. [0521] In some embodiments, the sense strand does not comprise a 2’-fluoro nucleotide in position opposite or complimentary to a thermally destabilizing modification of the duplex in the antisense strand. [0522] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro nucleotides.
  • the 2’-fluoro nucleotides can be located anywhere in the antisense strand.
  • the antisense strand can comprise a 2’-fluoro nucleotide at position 14, counting from 5’-end of the antisense strand.
  • the antisense comprises 2’-fluoro nucleotides at positions 2, 14 and 16, counting from the 5’-end of the antisense strand.
  • the antisense comprises 2’-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5’-end.
  • the antisense comprises 2’-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5’-end.
  • the antisense strand comprises at least one 2’-fluoro nucleotide adjacent to a destabilizing modification.
  • the 2’-fluoro nucleotide can be the nucleotide at the 5’-end or the 3’-end of a destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification.
  • the antisense strand comprises a 2’-fluoro nucleotide at each of the 5’-end and the 3’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the antisense strand comprises at least two 2’-fluoro nucleotides at the 3’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • both the sense and the antisense strands comprise at least one 2’-fluoro nucleotide.
  • the 2’-fluoro modification can occur on any nucleotide of the sense strand or antisense strand.
  • the 2’-fluoro modification can occur on every nucleotide on the sense strand and/or antisense strand; each 2’-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2’- fluoro modifications in an alternating pattern.
  • the alternating pattern of the 2’-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2’-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2’-fluoro modifications on the antisense strand.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides.
  • the 2’-OMe nucleotides all can be present in one strand.
  • the 2’-OMe nucleotide may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’- OMe nucleotides.
  • the 2’-OMe nucleotides can be located anywhere in the sense strand.
  • the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides.
  • the 2’-OMe nucleotides can be located anywhere in the antisense strand.
  • the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-deoxy, e.g., 2’-H ribose nucleotides.
  • the dsRNA can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 102’-deoxy, e.g., 2’-H nucleotides.
  • the 2’-deoxy nucleotide may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the dsRNA can comprise at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2’-deoxy modifications in a central region of the sense strand and/or the antisense strand.
  • At least one of the sense stand and the antisense can comprise at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven or more, 2’-deoxy modification in positions 5-17, e.g., positions 6-16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5’-end of the sense strand or the antisense strand.
  • the antisense strand comprises 1, 2, 3, 4, 5 or 6 of 2’-deoxy nucleotides.
  • antisense strand can comprise 2, 3, 4, 5 or 6 of 2’-deoxy nucleotides.
  • the 2’-deoxy nucleotides can be located anywhere in the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at 1, 2, 3 or 4 of positions 2, 5, 7, and 12, counting from 5’-end of the antisense strand.
  • the antisense comprises a 2’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’- deoxy nucleotide at positions 5, 7 and 12, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5 and 7, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7 and 12, counting from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7, 12 and 14, counting, from 5’-end of the antisense strand.
  • the antisense strand comprises a 2’- deoxy nucleotide at positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the antisense strand [0531]
  • the antisense comprises a 2’-deoxy nucleotide at position 2 or 12, counting from 5’-end of the antisense strand.
  • the antisense comprises a 2’-deoxy nucleotide at position 12, counting from 5’-end of the antisense strand.
  • the dsRNA comprises at least three 2’-deoxy modifications, wherein the 2’-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5’-end of the antisense strand, and at position 11 of the sense strand, counting from 5’-end of the sense strand.
  • the dsRNA comprises at least five 2’-deoxy modifications, wherein the 2’-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5’-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5’- end of the sense strand.
  • the dsRNA comprises at least seven 2’-deoxy modifications, wherein the 2’-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5’-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5’-end of the sense strand.
  • the antisense strand comprises at least five 2’-deoxy modifications at positions 2, 5, 7, 12 and 14, counting from 5’-end of the antisense strand.
  • the sense strand does not comprise a 2’-deoxy nucleotide at position 11, counting from 5 ’-end of the sense strand.
  • the dsRNA can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising a non-natural nucleobase
  • a nucleotide comprising a non-natural nucleobase can be present anywhere in the dsRNA molecule.
  • a nucleotide comprising a non-natural nucleobase can be present in the sense strand or a nucleotide comprising a non-natural nucleobase can be present in the antisense strand.
  • two or more nucleotides comprising a non-natural nucleobase are present in the dsRNA molecule, they can all be in the sense strand, antisense strand or both in the sense and antisense strands.
  • the dsRNA molecule described herein can further comprise at least one phosphorothioate or methylphosphonate intemucleoside linkage.
  • the phosphorothioate or methylphosphonate intemucleoside linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the intemucleoside linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each intemucleoside linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both intemucleoside linkage modifications in an alternating pattern.
  • the alternating pattern of the intemucleoside linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the intemucleoside linkage modification on the sense strand may have a shift relative to the alternating pattern of the intemucleoside linkage modification on the antisense strand.
  • the dsRNA molecule comprises the phosphorothioate or methylphosphonate intemucleoside linkage modification in the overhang region.
  • the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate intemucleoside linkage between the two nucleotides.
  • Intemucleoside linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region.
  • the overhang nucleotides may be linked through phosphorothioate or methylphosphonate intemucleoside linkage, and optionally, there may be additional phosphorothioate or methylphosphonate intemucleoside linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide.
  • the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3, 4, 5 or 6 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleoside linkages separated by 1, 2, 3 or 4 phosphate internucleoside linkages, wherein one of the phosphorothioate or methylphosphonate internucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.
  • the dsRNA molecule described herein further comprises one or more phosphorothioate or methylphosphonate internucleoside linkage modification within 1-10 of the termini position(s) of the sense and/or antisense strand.
  • one or more phosphorothioate or methylphosphonate internucleoside linkage modification within 1-10 of the termini position(s) of the sense and/or antisense strand.
  • at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleoside linkage at one end or both ends of the sense and/or antisense strand.
  • the dsRNA molecule described herein comprises one or more phosphorothioate or methylphosphonate internucleoside linkage modification within 1-10 of the internal region of the duplex of each of the sense and/or antisense strand.
  • at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleoside linkage at position 8-16 of the duplex region counting from the 5’-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleoside linkage modification within 1-10 of the termini position(s).
  • the dsRNA molecule described herein further comprises one to five phosphorothioate or methylphosphonate internucleoside linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleoside linkage modification(s) within the last 3 positions of the sense strand (counting from the 5’-end), and one to five phosphorothioate or methylphosphonate internucleoside linkage modification at positions 1 and 2 and one to five phosphorothioate or methylphosphonate internucleoside linkage modification within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate internucleoside linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleoside linkage modification within the last six positions of the sense strand (counting from the 5’-end), and one phosphorothioate internucleoside linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleoside linkage modifications within the last six the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications within position 1-5 and one phosphorothioate internucleoside linkage modification within the last six positions of the sense strand (counting from the 5’-end), and one phosphorothioate internucleoside linkage modification at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications within position 1-5 and two phosphorothioate internucleoside linkage modifications within the last four positions of the sense strand (counting from the 5’-end), and one phosphorothioate internucleoside linkage modification at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications within position 1-5 and two phosphorothioate internucleoside linkage modifications within the last four positions of the sense strand (counting from the 5’-end), and one phosphorothioate internucleoside linkage modification at positions 1 and 2 and one phosphorothioate internucleoside linkage modification within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate internucleoside linkage modification within position 1-5 and one phosphorothioate internucleoside linkage modification within the last four positions of the sense strand (counting from the 5’-end), and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate internucleoside linkage modification within position 1-5 and one within the last six positions of the sense strand (counting from the 5’-end), and two phosphorothioate internucleoside linkage modification at positions 1 and 2 and one phosphorothioate internucleoside linkage modification within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate internucleoside linkage modification within position 1-5 (counting from the 5’- end) of the sense strand, and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and one phosphorothioate internucleoside linkage modification within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications within position 1-5 (counting from the 5’- end) of the sense strand, and one phosphorothioate internucleoside linkage modification at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications within position 1-5 and one within the last six positions of the sense strand (counting from the 5’-end), and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and one phosphorothioate internucleoside linkage modification within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications within position 1-5 and one phosphorothioate internucleoside linkage modification within the last six positions of the sense strand (counting from the 5’-end), and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications within position 1-5 and one phosphorothioate internucleoside linkage modification within the last six positions of the sense strand (counting from the 5’-end), and one phosphorothioate internucleoside linkage modification at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications at position 1 and 2, and two phosphorothioate internucleoside linkage modifications at position 20 and 21 of the sense strand (counting from the 5’-end), and one phosphorothioate internucleoside linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate internucleoside linkage modification at position 1, and one phosphorothioate internucleoside linkage modification at position 21 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleoside linkage modifications at positions 1 and 2 and two phosphorothioate internucleoside linkage modifications at positions 20 and 21 the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate internucleoside linkage modifications at position 1 and 2, and two phosphorothioate internucleoside linkage modifications at position 21 and 22 of the sense strand (counting from the 5’-end), and one phosphorothioate internucleoside linkage modification at positions 1 and one phosphorothioate internucleoside linkage modification at position 21 of the antisense strand (counting from the 5’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate internucleoside linkage modification at position 1, and one phosphorothioate intemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 21 and 22 the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications at position 1 and 2, and two phosphorothioate intemucleoside linkage modifications at position 22 and 23 of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and one phosphorothioate intemucleoside linkage modification at position 21 of the antisense strand (counting from the 5 ’-end).
  • the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification at position 1, and one phosphorothioate intemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 22 and 23 the antisense strand (counting from the 5 ’-end).
  • the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand.
  • the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the sense strand.
  • the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’ -end of the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the antisense strand.
  • the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’ end of the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides n and n-1, and between nucleotides n-1 and n-2, where n is length of the antisense strand, i.e, number of nucleotides in the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the antisense strand.
  • the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’ -end of the antisense strand and at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the antisense strand.
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5’-end of the antisense strand and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3’-end of the antisense strand.
  • the sense strand comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand and the antisense strand comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 5’-end of the antisense strand.
  • the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5’-end of the sense strand
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5’-end of the antisense strand.
  • the sense strand comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand and the antisense strand comprises at least two phosphorothioate internucleoside linkages between the first five nucleotides counting from the 3’-end of the antisense strand.
  • the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5’-end of the sense strand
  • the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3’-end of the antisense strand.
  • dsRNA molecule described herein comprises a pattern of backbone chiral centers.
  • a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration.
  • a common pattern of backbone chiral centers comprises at least 16 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages in the Rp configuration.
  • a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration, and no more than 8 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral.
  • a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral.
  • the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous.
  • the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous.
  • the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.
  • dsRNA molecule described herein comprises a block is a stereochemistry block.
  • a block is an Rp block in that each internucleotidic linkage of the block is Rp.
  • a 5’-block is an Rp block.
  • a 3’-block is an Rp block.
  • a block is an Sp block in that each internucleotidic linkage of the block is Sp.
  • a 5’-block is an Sp block.
  • a 3’-block is an Sp block.
  • provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks.
  • provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.
  • dsRNA molecule described herein comprises a 5’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 5’-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’- block is an Sp block wherein each of internucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification.
  • a 5’-block comprises 4 or more nucleoside units.
  • a 5’-block comprises 5 or more nucleoside units.
  • a 5’-block comprises 6 or more nucleoside units.
  • a 5’-block comprises 7 or more nucleoside units.
  • a 3’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification.
  • a 3’-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3’-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3’-block comprises 4 or more nucleoside units. In some embodiments, a 3’-block comprises 5 or more nucleoside units. In some embodiments, a 3’-block comprises 6 or more nucleoside units.
  • a 3’-block comprises 7 or more nucleoside units.
  • dsRNA molecule described herein comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc.
  • A is followed by Sp.
  • Rp In some embodiments, A is followed by natural phosphate linkage (PO).
  • U is followed by Sp.
  • U is followed by Rp.
  • U is followed by natural phosphate linkage (PO).
  • C is followed by Sp.
  • C is followed by Rp.
  • C is followed by natural phosphate linkage (PO).
  • G is followed by Sp.
  • G is followed by Rp.
  • G is followed by natural phosphate linkage (PO).
  • C and U are followed by Sp.
  • C and U are followed by Rp.
  • C and U are followed by natural phosphate linkage (PO).
  • a and G are followed by Sp.
  • a and G are followed by Rp.
  • the dsRNA molecule described herein comprises one or more overhang regions and/or capping groups of dsRNA molecule at the 3’-end, or 5’-end or both ends of a strand.
  • the overhang can be 1-10 nucleotides in length.
  • the overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length.
  • the overhang is 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length.
  • the overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.
  • the overhang can form a mismatch with the target sequence or it can be complementary to the gene sequences being targeted or it can be the other sequence.
  • the first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
  • the nucleotides in the overhang region of the dsRNA molecule described herein can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-Fluoro 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl- 5-methyluridine, 2’-O-methoxyethyladenosine, 2’-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h’GNA, and any combinations thereof.
  • 2’-sugar modified such as, 2’-Fluoro 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl- 5-methyluridine, 2’-O-methoxyethyladenosine, 2’-O-methoxyethyl-5-methylcy
  • dTdT can be an overhang sequence for either end on either strand.
  • the overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.
  • the 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule described herein may be phosphorylated.
  • the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different.
  • the overhang is present at the 3’-end of the sense strand, antisense strand or both strands.
  • this 3’-overhang is present in the antisense strand. In some embodiments, this 3’-overhang is present in the sense strand.
  • the dsRNA molecule described herein may comprise only a single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall stability.
  • the single-stranded overhang is located at the 3'-terminal end of the sense strand or, alternatively, at the 3'-terminal end of the antisense strand.
  • the dsRNA can also have a blunt end, located at the 5’-end of the antisense strand (or the 3’-end of the sense strand) or vice versa.
  • the antisense strand of the dsRNA has a nucleotide overhang at the 3’-end, and the 5’-end is blunt. While not bound by theory, the asymmetric blunt end at the 5’-end of the antisense strand and 3’-end overhang of the antisense strand favor the guide strand loading into RISC process.
  • the single overhang is at least one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in length.
  • the dsRNA has a 2 nucleotide overhang on the 3’-end of the antisense strand and a blunt end at the 5’-end of the antisense strand.
  • the dsRNA described herein can comprise one or more modified nucleotides. For example, every nucleotide in the sense strand and antisense strand of the dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar; replacement of the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.
  • nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not.
  • a modification may only occur at a 3’ or 5’ terminal position, may only occur in a central region, may only occur at a non-terminal region, or may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand.
  • a modification may occur in a double strand region, a single strand region, or in both.
  • a modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA.
  • a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini.
  • the 5’ end or ends can be phosphorylated.
  • purine nucleotides in overhangs.
  • all or some of the bases in a 3’ or 5’ overhang may be modified, e.g., with a modification described herein.
  • Modifications can include, e.g., the use of modifications at the 2’ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2’-deoxy-2’-fluoro (2’-F) or 2’-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications.
  • the dsRNA molecule described herein comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1’, B2’, B3’, B4’ regions.
  • alternating motif or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand.
  • the alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern.
  • the alternating motif can be “ABABABABABAB...,” “AABBAABBAABB...,” “AABAABAABAAB...,” “AAABAAABAAAB...,” “AAABBBAAABBB...,” or “ABCABCABCABC...,” etc.
  • the type of modifications contained in the alternating motif may be the same or different.
  • the alternating pattern i.e., modifications on every other nucleotide
  • the alternating pattern may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB...”, “ACACAC...” “BDBDBD...” or “CDCDCD...,” etc.
  • the dsRNA molecule described herein comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted.
  • the shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa.
  • the sense strand when paired with the antisense strand in the dsRNA duplex the alternating motif in the sense strand may start with “ABABAB” from 5’-3’ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3’-5’of the strand within the duplex region.
  • the alternating motif in the sense strand may start with “AABBAABB” from 5’-3’ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3’-5’of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.
  • the oligonucleotides described herein or at least one e.g., both strand of a dsRNA described herein are 5’ phosphorylated or include a phosphoryl analog at the 5’ prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5'- monophosphate ((HO) 2 (O)P-O-5'); 5'-diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O- 5'); 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)P-O-5'); 5'
  • 5'- alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO) 2 (O)P-NH-5', (HO)(NH 2 )(O)P-O-5'), 5'-alkylphosphonates (e.g., RP(OH)(O)-O-5'-, R alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonates (i.e.
  • exemplary 5’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO) 2 (X)P-O[-(CH 2 ) a -O-P(X)(OH)-O] b - 5', ((HO)2(X)P-O[-(CH 2 ) a -P(X)(OH)-O] b - 5', ((HO)2(X)P-[-(CH 2 ) a -P(X)(OH)-O] b - 5', ((HO)2(X)P-[-(CH 2 ) a -O-P(X)(OH)-O] b - 5'; dialkyl
  • the oligonucleotide or at least one (e.g., both) strand of a dsRNA described herein comprises a 5’-vinylphosphonate group.
  • the oligonucleotide or at least one (e.g., both) strand of a dsRNA described herein comprises a 5’-E-vinyl or at least one (e.g., both) strand of a dsRNA described herein phosphonate group.
  • the oligonucleotide comprises a 5’-Z- vinylphosphonate group.
  • the 5’-modification can be placed in the antisense strand of a double- stranded nucleic acid, e.g., dsRNA molecule.
  • the antisense comprises a 5’-E- vinylphosphonate.
  • the antisense strand comprises a 5’-Z- vinylphosphonate group.
  • the sense strand comprises a 5’-morpholino, a 5’- dimethylamino, a 5’-deoxy, an inverted abasic, or an inverted abasic locked nucleic acid modification at the 5’-end.
  • the dsRNA agents of the invention can comprise thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5’-end of the antisense strand) to reduce or inhibit off-target gene silencing.
  • dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5’ end, of the antisense strand have reduced off-target gene silencing activity.
  • the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5’ region of the antisense strand.
  • thermally destabilizing modification of the duplex is located in positions 2-9, or preferably positions 4-8, from the 5’-end of the antisense strand.
  • the thermally destabilizing modification of the duplex is located at position 5, 6, 7 or 8 from the 5’-end of the antisense strand. [0597] In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5’-end of the antisense strand. [0598]
  • the dsRNA can also comprise one or more stabilizing modifications.
  • the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand.
  • both the sense and the antisense strands comprise at least two stabilizing modifications.
  • the stabilizing modification can occur on any nucleotide of the sense strand or antisense strand.
  • the stabilizing modification can occur on every nucleotide on the sense strand and/or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern.
  • the alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.
  • the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • a stabilizing modification in the antisense strand can be present at any positions.
  • the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16 from the 5’-end.
  • the antisense comprises stabilizing modifications at positions 2, 6, 14 and 16 from the 5’-end.
  • the antisense comprises stabilizing modifications at positions 2, 14 and 16 from the 5’-end.
  • the antisense comprises at least one stabilizing modification adjacent to the destabilizing modification.
  • the stabilizing modification can be the nucleotide at the 5’-end or the 3’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification.
  • the antisense strand comprises a stabilizing modification at each of the 5’-end and the 3’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.
  • the antisense strand comprises at least two stabilizing modifications at the 3’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.
  • the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.
  • a stabilizing modification in the sense strand can be present at any positions.
  • the sense strand comprises stabilizing modifications at positions 7, 10 and 11 from the 5’-end.
  • the sense strand comprises stabilizing modifications at positions 7, 9, 10 and 11 from the 5’-end.
  • the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5’-end of the antisense strand.
  • the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5’-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four stabilizing modifications. [0602] In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand. [0603] It is noted a thermally stabilizing modification can replace a 2’-fluoro nucleotide in the sense and/or antisense strand.
  • a 2’-fluoro nucleotide at positions 8, 9, 10, 11 and/or 12, counting from 5’-end, of the sense strand can be replaced with a thermally stabilizing modification.
  • a 2’-fluoro nucleotide at position 14, counting from 5’-end, of the antisense strand can be replaced with a thermally stabilizing modification.
  • At least 40% for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 5 after in vivo administration.
  • at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 6 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 7 after in vivo administration.
  • at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 8 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 9 after in vivo administration. In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 10 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 11 after in vivo administration. In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 12 after in vivo administration.
  • At least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 13 after in vivo administration. In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 14 after in vivo administration.
  • At least 40% for example at least 45%, at least 50%, at least 55%, at least 60%., at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 15 after in vivo administration.
  • the oligonucleotide described herein or the antisense strand of the dsRNA molecule described herein comprises a nucleotide sequence substantially complementary to a target nucleic acid, e.g., a target gene or mRNA.
  • the disclosure is directed to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene.
  • the present invention further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in vitro.
  • the disclosure is directed to a use of an oligonucleotide and/or dsRNA molecule described herein for use in inhibiting expression of a target gene in a subject.
  • the subject may be any animal, such as a mammal, e.g., a mouse, a rat, a sheep, a cattle, a dog, a cat, or a human
  • the oligonucleotide and/or dsRNA molecule described herein is administered in buffer.
  • oligonucleotide and/or dsRNA molecule described herein described herein can be formulated for administration to a subject.
  • a formulated oligonucleotide and/or dsRNA composition can assume a variety of states.
  • the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water).
  • the siRNA is in an aqueous phase, e.g., in a solution that includes water.
  • the aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • a delivery vehicle e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition).
  • the siRNA composition is formulated in a manner that is compatible with the intended method of administration, as described herein.
  • the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
  • a oligonucleotide and/or dsRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide and/or dsRNA, e.g., a protein that complexes with oligonucleotide and/or dsRNA.
  • another agent e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide and/or dsRNA, e.g., a protein that complexes with oligonucleotide and/or dsRNA.
  • Still other agents include chelating agents, e.g., EDTA (e.g., to remove divalent cations such as Mg 2+ ), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
  • the oligonucleotide and/or dsRNA preparation includes another dsRNA compound, e.g., a second dsRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different siRNA species. Such dsRNAs can mediate RNAi with respect to a similar number of different genes.
  • the oligonucleotide and/or dsRNA preparation includes at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA).
  • a oligonucleotide and/or dsRNA composition for the treatment of a viral disease might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor).
  • a dsRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.
  • exemplary formulations which can be used for administering the oligonucleotide and/or dsRNA according to the present invention are discussed below.
  • Liposomes Liposomes.
  • a oligonucleotide and/or dsRNA preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle.
  • liposome refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the oligonucleotide and/or dsRNA composition.
  • the lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the oligonucleotide and/or dsRNA composition, although in some examples, it may.
  • Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the oligonucleotide and/or dsRNA are delivered into the cell where the dsRNA can specifically bind to a target RNA and can mediate RNAi.
  • the liposomes are also specifically targeted, e.g., to direct the oligonucleotide and/or dsRNA to particular cell types.
  • a liposome containing oligonucleotide and/or dsRNA can be prepared by a variety of methods.
  • the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component.
  • the lipid component can be an amphipathic cationic lipid or lipid conjugate.
  • the detergent can have a high critical micelle concentration and may be nonionic.
  • Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine.
  • the dsRNA preparation is then added to the micelles that include the lipid component.
  • the cationic groups on the lipid interact with the siRNA and condense around the dsRNA to form a liposome.
  • the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of oligonucleotide and/or dsRNA.
  • a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition.
  • the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favor condensation.
  • a polymer other than a nucleic acid e.g., spermine or spermidine. pH can also be adjusted to favor condensation.
  • WO 96/37194 Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413- 7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No.
  • lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, which is incorporated by reference in its entirety).
  • Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984, which is incorporated by reference in its entirety). These methods are readily adapted to packaging oligonucleotide and/or dsRNA preparations into liposomes.
  • Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated by reference in its entirety).
  • liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Examples of other methods to introduce liposomes into cells in vitro include U.S. Pat. No.5,283,185; U.S. Pat. No.5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem.269:2550, 1994; Nabel, Proc. Natl. Acad. Sci.90:11307, 1993; Nabel, Human Gene Ther.3:649, 1992; Gershon, Biochem.32:7143, 1993; and Strauss EMBO J.11:417, 1992.
  • cationic liposomes are used.
  • Cationic liposomes possess the advantage of being able to fuse to the cell membrane.
  • Non-cationic liposomes although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver siRNAs to macrophages.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated siRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245).
  • Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
  • a positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of siRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat.
  • siRNA see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat.
  • a DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles.
  • DOTAP 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane
  • LipofectinTM Bethesda Research Laboratories, Gaithersburg, Md. is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes.
  • DOTAP 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane
  • cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (TransfectamTM, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No.5,171,678).
  • DOGS 5-carboxyspermylglycine dioctaoleoylamide
  • DPES dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide
  • Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991, which is incorporated by reference in its entirety).
  • these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions.
  • Other commercially available cationic lipid products include DMRIE and DMRIE- HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland).
  • DOSPA Lipofectamine
  • Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
  • Liposomal formulations are particularly suited for topical administration. Liposomes present several advantages over other formulations.
  • liposomes are used for delivering siRNA to epidermal cells and also to enhance the penetration of siRNA into dermal tissues, e.g., into skin.
  • the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J.
  • Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol.
  • Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/ cholesterol/polyoxyethylene- 10-stearyl ether) were used to deliver a drug into the dermis of mouse skin.
  • Such formulations with dsRNA descreibed herein are useful for treating a dermatological disorder.
  • Liposomes that include oligonucleotide and/or dsRNA described herein can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome.
  • transfersomes are a type of deformable liposomes. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include oligonucleotide and/or dsRNA described herein can be delivered, for example, subcutaneously by infection in order to deliver dsRNA to keratinocytes in the skin.
  • lipid vesicles In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transfersomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), selfrepairing, and can frequently reach their targets without fragmenting, and often self-loading. [0631] Other formulations amenable to the present invention are described in United States provisional application serial nos.
  • the oligonucleotide and/or dsRNA compositions can include a surfactant.
  • the dsRNA is formulated as an emulsion that includes a surfactant.
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class. [0634] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps. [0635] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. [0636] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. [0637] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, NY, 1988, p.285).
  • mice and other Membranous Formulations are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
  • a mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the oligonucleotide and/or dsRNA composition, an alkali metal C 8 to C 22 alkyl sulphate, and a micelle forming compounds.
  • Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyl oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.
  • the micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.
  • a first micellar composition is prepared which contains the oligonucleotide and/or dsRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition.
  • the micellar composition is prepared by mixing the dsRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
  • Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth.
  • phenol and/or m-cresol may be added with the micelle forming ingredients.
  • An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
  • the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant.
  • Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.
  • dsRNA preparations can be incorporated into a particle, e.g., a microparticle.
  • Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.
  • Pharmaceutical compositions [0646] The oligonucleotide and/or dsRNA described herein can be formulated for pharmaceutical use.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the oligonucleotide and/or dsRNA described herein.
  • Pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the dsRNA molecules in any of the preceding embodiments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.
  • the phrase “therapeutically-effective amount” as used herein means that amount of a compound, material, or composition comprising a dsRNA molecule described herein which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (1
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention.
  • an aforementioned formulation renders orally bioavailable a compound of the present invention.
  • Methods of preparing these formulations or compositions include the step of bringing into association an oligonucleotide and/or dsRNA with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • the oligonucleotide and/or dsRNA described herein may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
  • treatment is intended to encompass therapy and cure.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • the oligonucleotide and/or dsRNA described herein or a pharmaceutical composition comprising an oligonucleotide and/or dsRNA described herein can be administered to a subject using different routes of delivery.
  • a composition that includes an oligonucleotide and/or dsRNA described herein described herein can be delivered to a subject by a variety of routes.
  • routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
  • the oligonucleotide and/or dsRNA described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
  • the route and site of administration may be chosen to enhance targeting.
  • Lung cells might be targeted by administering the oligonucleotide and/or dsRNA described herein in aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the oligonucleotide and/or dsRNA described herein and mechanically introducing the oligonucleotide and/or dsRNA described herein.
  • a method of administering an oligonucleotide and/or dsRNA described herein, to a subject e.g., a human subject.
  • the present invention relates to an oligonucleotide and/or dsRNA described herein for use in inhibiting expression of a target gene in a subject.
  • the method or the medical use includes administering a unit dose of the oligonucleotide and/or dsRNA described herein.
  • the unit dose is less than 10 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g., about 4.4 x 10 16 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of oligonucleotide and/or dsRNA described herein per kg of bodyweight.
  • RNA agent e.g., about 4.4 x 10 16 copies
  • the defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target gene.
  • the unit dose for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application. In some embodiments dosages may be less than 10, 5, 2, 1, or 0.1 mg/kg of body weight. [0662] In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.
  • the effective dose is administered with other traditional therapeutic modalities.
  • a subject is administered an initial dose and one or more maintenance doses.
  • the maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half less of the initial dose.
  • a maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 ⁇ g to 15 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day.
  • the maintenance doses are, for example, administered no more than once every 2, 5, 10, or 30 days.
  • the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient.
  • the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days.
  • the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state.
  • the dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
  • the effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • a delivery device e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
  • the composition includes a plurality of dsRNA molecule species.
  • the dsRNA molecule species has sequences that are non- overlapping and non-adjacent to another species with respect to a naturally occurring target sequence.
  • the plurality of dsRNA molecule species is specific for different naturally occurring target genes.
  • the dsRNA molecule is allele specific.
  • the oligonucleotide and/or dsRNA described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.
  • the administration of the oligonucleotide and/or dsRNA composition described herein is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • the invention provides methods, compositions, and kits, for rectal administration or delivery of oligonucleotide and/or dsRNA composition described herein. Methods of inhibiting expression of a target gene [0670] Aspects of the disclosure also relate to methods for inhibiting the expression of a target gene.
  • the method comprises administering to the subject in an amount sufficient to inhibit expression of the target gene: (i) a double-stranded RNA described herein, where the wherein the first strand is complementary to a target gene; and/or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to a target gene.
  • the present disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell.
  • the present disclosure further relates to a use of an oligonucleotide and/or dsRNA molecule described herein for inhibiting expression of a target gene in a target cell in vitro.
  • the invention relates to a method of modulating the expression of a target gene in a cell, comprising administering to said cell an oligonucleotide and/or dsRNA molecule described herein.
  • the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene,
  • Embodiment A An oligonucleotide of the formula, wherein: R is hydroxyl, protected hydroxyl, or a 2’-ribose modification (e.g., hydrogen, methoxy, halogen, fluoro, 2-methoxyethoxy, C 1-30 alkyl, or C 1-30 alkoxy such as n-hexadecyloxy), -O-C 4- 30 alkyl-ON(CH 2 R1)(CH 2 R1), or -O-C 4-30 alkyl-ON(CH 2 R1)(CH 2 R2); each R1 and R2 is independently a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, C 1-30 alkyl, C 1-30 alkenyl, or C 1-30 alkynyl, each optionally substituted with one or more aldehyde (-C(O)H),
  • R is hydroxyl, protected hydroxyl, or a 2’-ribose modification (e.
  • R is hydroxyl, protected hydroxyl, or a 2’-ribose modification (e.g., hydrogen, methoxy, halogen,fluoro, 2-methoxyethoxy, C 1-30 alkyl, or C 1-30 alkoxy such as n-hexadecyloxy), -O-C 4- 30 alkyl-ON(CH 2 R1)(CH 2 R1), or -O-C 4-30 alkyl-ON(CH 2 R1)(CH 2 R2); each R1 and R2 is independently a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, C 1-30 alkyl, C 1-30 alkenyl, or C 1-30 alkynyl, each optionally substituted with one or more aldehyde (-C(O)H), carboxylic acid (-COOH), C 1-10 acyl (i.e., -C(O)C 1-10 alkyl), hydroxyl, halogen, cyano,
  • Embodiment C The oligonucleotide of Embodiment A or B, wherein R is hydrogen, methoxy, halogen, fluoro, 2-methoxyethoxy, C 1-30 alkyl, or C 1-30 alkoxy (such as n- hexadecyloxy).
  • Embodiment D The oligonucleotide of any one of Embodiments A-C, wherein R is hydrogen, methoxy, fluoro, or 2-methoxyethoxy.
  • Embodiment E The oligonucleotide of any one of Embodiments A-D, wherein R is hydrogen.
  • Embodiment F The oligonucleotide of any one of Embodiments A-D, wherein R is methoxy.
  • Embodiment G The oligonucleotide of any one of Embodiments A-D, wherein R is fluoro.
  • Embodiment H The oligonucleotide of any one of Embodiments A-D, wherein R is methoxyethoxy.
  • Embodiment I The oligonucleotide of any one of Embodiments A-H, wherein R1 is a targeting ligand and R2 is a pharmacokinetic modifier.
  • Embodiment JA double-stranded RNA comprising 20 – 100 nucleotides, wherein: the first strand is according to any one of Embodiments A-I, and the second strand optionally comprises 1 or more modified nucleotides and is complementary to the first strand, wherein Hone or both strands has a 1 – 5 nucleotide overhang on its respective 5’-end or 3’-end, or only one strand has a 2 nucleotide overhang on its 5’-end or 3’-end, or only one strand has a 2 nucleotide overhand on its 3’-end.
  • Embodiment K A method of reducing the expression of a target gene in a subject (e.g., human), comprising administering to the subject either: (i) a double-stranded RNA according to Embodiment J, wherein the first strand or the second strand is complementary to a target gene; or (ii) an oligonucleotide according to any one of Embodiments A-I, wherein the oligonucleotide is complementary to a target gene.
  • Embodiment L A compound of the formula, wherein: R is hydroxyl, protected hydroxyl, or a 2’-ribose modification (e.g., hydrogen, methoxy, halogen,fluoro, 2-methoxyethoxy, C 1-30 alkyl, or C 1-30 alkoxy such as n-hexadecyloxy), -O-C 4- 30 alkyl-ON(CH 2 R1)(CH 2 R1), or -O-C 4-30 alkyl-ON(CH 2 R1)(CH 2 R2); each R1 and R2 is independently a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, C 1-30 alkyl, C 1-30 alkenyl, or C 1-30 alkynyl, each optionally substituted with one or more aldehyde (-C(O)H), carboxylic acid (-COOH), C 1-10 acyl (i.e., -C(O)C 1-10
  • Embodiment M The compound of Embodiment L, wherein R is hydrogen, methoxy, halogen, fluoro, 2-methoxyethoxy, C 1-30 alkyl, or C 1-30 alkoxy (such as n-hexadecyloxy).
  • Embodiment N The compound of any one of Embodiments L-M, wherein R is hydrogen, methoxy, fluoro, or 2-methoxyethoxy.
  • Embodiment O The compound of any one of Embodiments L-N, wherein R is hydrogen.
  • Embodiment P The compound of any one of Embodiments L-N, wherein R is methoxy.
  • Embodiment Q The compound of any one of Embodiments L-N, wherein R is fluoro.
  • Embodiment R The compound of any one of Embodiments L-N, wherein R is methoxyethoxy.
  • Embodiment S The compound of any one of Embodiments L-R, wherein R1 is a targeting ligand and R2 is a pharmacokinetic modifier.
  • alkyl refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain.
  • “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms.
  • the alkyl group can be optionally substituted with one or more alkyl group substituents which can be the same or different, where “alkyl group substituent” includes halo, amino, aryl, hydroxyl, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl.
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.
  • exemplary alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl.
  • Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.
  • a “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH 2 group to an NH group or an O group).
  • the term “heteroalkyl” include optionally substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof.
  • the heteroatom(s) is placed at any interior position of the heteroalkyl group.
  • alkenyl refers to an alkyl group containing at least one carbon-carbon double bond.
  • the alkenyl group can be optionally substituted with one or more “alkyl group substituents.”
  • Exemplary alkenyl groups include vinyl, allyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-1-yl and heptadec-8,11-dien-1-yl.
  • alkynyl refers to an alkyl group containing a carbon-carbon triple bond.
  • the alkynyl group can be optionally substituted with one or more “alkyl group substituents.”
  • Exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl.
  • Useful alkynyl groups include the lower alkynyl groups.
  • cycloalkyl refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms. The cycloalkyl group can be optionally partially unsaturated.
  • the cycloalkyl group can be also optionally substituted with an aryl group substituent, oxo and/or alkylene.
  • Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl.
  • Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.
  • Heterocyclyl refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively).
  • Cxheterocyclyl and Cx-Cyheterocyclyl are typically used where X and Y indicate the number of carbon atoms in the ring system.
  • 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent.
  • exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4- morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3- dioxanyl, 1,4-dioxanyland the like.
  • Aryl refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms.
  • the aryl group can be optionally substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and —NRR', where R and R' are each independently hydrogen, alkyl, aryl and aralkyl.
  • aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl.
  • “Heteroaryl” refers to an aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.
  • O, N, or S e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.
  • Exemplary aryl and heteroaryls include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzisothi
  • halogen refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen radioisotope or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.
  • halogen-substituted moiety or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.
  • haloalkyl refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different.
  • fluoroalkyl and fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.
  • exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (C 1 -C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF 3 ), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like).
  • amino means -NH 2 .
  • alkylamino means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., –NH(alkyl).
  • dialkylamino means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals attached to the nitrogen, e.g., –N(alkyl)(alkyl).
  • alkylamino includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.”
  • arylamino means a nitrogen moiety having at least one aryl radical attached to the nitrogen. For example, -NHaryl, and —N(aryl) 2 .
  • heteroarylamino means a nitrogen moiety having at least one heteroaryl radical attached to the nitrogen. For example —NHheteroaryl, and —N(heteroaryl) 2 .
  • two substituents together with the nitrogen can also form a ring.
  • the compounds described herein containing amino moieties can include protected derivatives thereof.
  • Suitable protecting groups for amino moieties include acetyl, tertbutoxycarbonyl, benzyloxycarbonyl, and the like.
  • Exemplary alkylamino includes, but is not limited to, NH(C 1 - C 10 alkyl), such as —NHCH 3 , —NHCH 2 CH 3 , —NHCH 2 CH 2 CH 3 , and —NHCH(CH 3 ) 2 .
  • Exemplary dialkylamino includes, but is not limited to, —N(C 1 -C 10 alkyl) 2 , such as N(CH 3 ) 2 , —N(CH 2 CH 3 ) 2 , —N(CH 2 CH 2 CH 3 ) 2 , and —N(CH(CH 3 ) 2 ) 2 .
  • aminoalkyl means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms (—N—) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl.
  • an (C 2 -C 6 ) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.
  • hydroxyl and hydroxyl mean the radical —OH.
  • alkoxyl or alkoxy refers to an alkyl group, as defined above, having an oxygen radical attached thereto, and can be represented by one of -O-alkyl, -O- alkenyl, and -O-alkynyl.
  • Aroxy can be represented by –O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein.
  • alkoxy and aroxy groups can be substituted as described above for alkyl.
  • exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n- propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O- hexyl, O- cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.
  • carbonyl means the radical —C(O)—.
  • carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.
  • carboxy means the radical —C(O)O—.
  • compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like.
  • a carboxy group includes —COOH, i.e., carboxyl group.
  • cyano means the radical —CN.
  • nitro means the radical —NO 2 .
  • heteroatom refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, sulfur and halogens.
  • heteroatom moiety includes a moiety where the atom by which the moiety is attached is not a carbon.
  • alkylthio and “thioalkoxy” refer to an alkoxy group, as defined above, where the oxygen atom is replaced with a sulfur.
  • the “alkylthio” moiety is represented by one of -S-alkyl, -S-alkenyl, and -S-alkynyl.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • the term “alkylthio” also encompasses cycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.
  • Arylthio refers to aryl or heteroaryl groups.
  • sulfinyl means the radical —SO—.
  • the sulfinyl radical can be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, sulfoxides, and the like.
  • sulfonyl means the radical —SO 2 —. It is noted that the sulfonyl radical can be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids (-SO 3 H), sulfonamides, sulfonate esters, sulfones, and the like.
  • thiocarbonyl means the radical —C(S)—. It is noted that the thiocarbonyl radical can be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, thioketones, and the like.
  • thiocarbonyl refers to an alkyl-CO— group, wherein alkyl is as previously described.
  • Exemplary acyl groups comprise alkyl of 1 to about 30 carbon atoms. Exemplary acyl groups also include acetyl, propanoyl, 2-methylpropanoyl, butanoyl and palmitoyl.
  • Aroyl means an aryl-CO— group, wherein aryl is as previously described. Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.
  • Arylthio refers to an aryl-S— group, wherein the aryl group is as previously described. Exemplary arylthio groups include phenylthio and naphthylthio.
  • Aralkyl refers to an aryl-alkyl— group, wherein aryl and alkyl are as previously described. Exemplary aralkyl groups include benzyl, phenylethyl and naphthylmethyl.
  • Alkyloxy refers to an aralkyl-O— group, wherein the aralkyl group is as previously described.
  • An exemplary aralkyloxy group is benzyloxy.
  • Aralkylthio refers to an aralkyl-S— group, wherein the aralkyl group is as previously described.
  • An exemplary aralkylthio group is benzylthio.
  • Alkoxycarbonyl refers to an alkyl-O—CO— group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl.
  • Aryloxycarbonyl refers to an aryl-O—CO— group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.
  • Aralkoxycarbonyl refers to an aralkyl-O—CO— group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.
  • Carbamoyl refers to an H 2 N—CO— group.
  • Alkylcarbamoyl refers to a R'RN—CO— group, wherein one of R and R' is hydrogen and the other of R and R' is alkyl as previously described.
  • Dialkylcarbamoyl refers to R'RN—CO— group, wherein each of R and R' is independently alkyl as previously described.
  • Acyloxy refers to an acyl-O— group, wherein acyl is as previously described.
  • Acylamino refers to an acyl-NH— group, wherein acyl is as previously described.
  • Aroylamino refers to an aroyl-NH— group, wherein aroyl is as previously described.
  • substituted means that the specified group or moiety is unsubstituted or is substituted with one or more (typically 1, 2, 3, 4, 5 or 6 substituents) independently selected from the group of substituents listed below in the definition for “substituents” or otherwise specified.
  • substituted refers to a group “substituted” on a substituted group at any atom of the substituted group.
  • Suitable substituents include, without limitation, halogen, hydroxyl, caboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxylalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.
  • an optionally substituted group is substituted with 1 substituent. In some other embodiments, an optionally substituted group is substituted with 2 independently selected substituents, which can be same or different. In some other embodiments, an optionally substituted group is substituted with 3 independently selected substituents, which can be same, different or any combination of same and different. In still some other embodiments, an optionally substituted group is substituted with 4 independently selected substituents, which can be same, different or any combination of same and different. In yet some other embodiments, an optionally substituted group is substituted with 5 independently selected substituents, which can be same, different or any combination of same and different. [0743] An “isocyanato” group refers to a NCO group.
  • a “thiocyanato” group refers to a CNS group.
  • An “isothiocyanato” group refers to a NCS group.
  • dsRNA e.g., mRNA, e.g., a transcript of a gene that encodes a protein.
  • mRNA to be silenced is also referred to herein as mRNA to be silenced.
  • a gene is also referred to as a target gene.
  • the RNA to be silenced is an endogenous gene, exogenous gene or a pathogen gene.
  • RNAs other than mRNA e.g., tRNAs, and viral RNAs, can also be targeted.
  • the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target gene, e.g., mRNA.
  • silencing uses the RNAi machinery or process and a guide RNA, e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.
  • a guide RNA e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.
  • 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.
  • off-target and the phrase “off-target effects” refer to any instance in which an effector molecule against a given target causes an unintended affect by interacting either directly or indirectly with another target sequence, a DNA sequence or a cellular protein or other moiety.
  • an “off-target effect” may occur when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of an siRNA.
  • nucleoside means a glycosylamine comprising a nucleobase and a sugar.
  • Nucleosides includes, but are not limited to, naturally occurring nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups.
  • nucleotide refers to a glycosomine comprising a nucleobase and a sugar having a phosphate group covalently linked to the sugar. Nucleotides may be modified with any of a variety of substituents.
  • locked nucleic acid or “LNA” or “locked nucleoside” or “locked nucleotide” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system. Locked nucleic acids are also referred to as bicyclic nucleic acids (BNA).
  • BNA bicyclic nucleic acids
  • methyleneoxy LNA alone refers to ⁇ -D-methyleneoxy LNA.
  • the term “MOE” refers to a 2′-O-methoxyethyl substituent.
  • the term “gapmer” refers to a chimeric oligomeric compound comprising a central region (a “gap”) and a region on either side of the central region (the “wings”), wherein the gap comprises at least one modification that is different from that of each wing.
  • modifications include nucleobase, monomeric linkage, and sugar modifications as well as the absence of modification (unmodified).
  • the nucleotide linkages in each of the wings are different than the nucleotide linkages in the gap.
  • each wing comprises nucleotides with high affinity modifications and the gap comprises nucleotides that do not comprise that modification.
  • the nucleotides in the gap and the nucleotides in the wings all comprise high affinity modifications, but the high affinity modifications in the gap are different than the high affinity modifications in the wings.
  • the modifications in the wings are the same as one another. In certain embodiments, the modifications in the wings are different from each other.
  • nucleotides in the gap are unmodified and nucleotides in the wings are modified.
  • the modification(s) in each wing are the same. In certain embodiments, the modification(s) in one wing are different from the modification(s) in the other wing.
  • oligomeric compounds are gapmers having 2′-deoxynucleotides in the gap and nucleotides with high-affinity modifications in the wing.
  • BNA refers to bridged nucleic acid, and is often referred as constrained or inaccessible RNA.
  • BNA can contain a 5-, 6- membered, or even a 7-membered bridged structure with a “fixed” C 3 ’-endo sugar puckering.
  • the bridge is typically incorporated at the 2’-, 4’-position of the ribose to afford a 2’, 4’-BNA nucleotide (e.g., LNA, or ENA).
  • LNA refers to locked nucleic acid, and is often referred as constrained or inaccessible RNA.
  • LNA is a modified RNA nucleotide.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge (e.g., a methylene bridge or an ethylene bridge) connecting the 2′ hydroxyl to the 4′ carbon of the same ribose sugar.
  • the bridge can “lock” the ribose in the 3′-endo North) conformation: .
  • the term ‘ENA’ refers to ethylene-bridged nucleic acid, and is often referred as constrained or inaccessible RNA.
  • the “cleavage site” herein means the backbone linkage in the target gene or the sense strand that is cleaved by the RISC mechanism by utilizing the iRNA agent. And the target cleavage site region comprises at least one or at least two nucleotides on both side of the cleavage site. For the sense strand, the cleavage site is the backbone linkage in the sense strand that would get cleaved if the sense strand itself was the target to be cleaved by the RNAi mechanism.
  • the cleavage site can be determined using methods known in the art, for example the 5’-RACE assay as detailed in Soutschek et al., Nature (2004) 432, 173-178, which is incorporated by reference in its entirety.
  • the cleavage site region for a conical double stranded RNAi agent comprising two 21-nucleotides long strands wherein the strands form a double stranded region of 19 consecutive base pairs having 2-nucleotide single stranded overhangs at the 3’-ends
  • the cleavage site region corresponds to positions 9-12 from the 5’-end of the sense strand.
  • the terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g.
  • the absence of a given treatment can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • a “terminal region” of a strand refers to positions 1-4, e.g., positions 1, 2, 3, and 4, counting from the nearest end of the strand.
  • a 5’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 5’-end of the strand.
  • a 3’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 3’-end of the strand.
  • a 5’-terminal region for the antisense strand is positions 1, 2, 3 and 4 counting from the 5’-end of the antisense strand.
  • a preferred 5’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 5’-end of the antisense strand.
  • a 3’-terminal region for the antisense strand can be positions 1, 2, 3, and 4 counting from the 3’-end of the strand.
  • a preferred 3’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 3’- end of the antisense strand.
  • a 5’-terminal region for the sense strand is positions 1, 2, 3 and 4 counting from the 5’-end of the sense strand.
  • a preferred 5’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 5’-end of the sense strand.
  • a 3’-terminal region for the sense strand can be positions 1, 2, 3, and 4 counting from the 3’-end of the strand.
  • a preferred 3’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 3’-end of the sense strand.
  • a “central region” of a strand refers to positions 5-17, e.g., positions 6- 16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5’-end of the strand.
  • the central region of a strand means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the strand.
  • a preferred central region for the sense strand is positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5’-end of the sense strand.
  • a more preferred central region for the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5’-end of the sense strand.
  • a preferred central region for the antisense strand is positions 9, 10, 11, 12, 13, 14, 1516 and 17, counting from 5’-end of the antisense strand.
  • a more preferred central region for the antisense strand is positions 10, 11, 12, 13, 14, 15 and 16, counting from 5’- end of the antisense strand.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g. animal or a plant).
  • ex vivo refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube).
  • in vivo refers to events that occur within an organism (e.g. animal, plant, and/or microbe).
  • the term "subject" or "patient” refers to any organism to which a composition disclosed herein can be administered, e.g., for experimental, diagnostic, and/or therapeutic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • a subject can be male or female.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders.
  • compounds, compositions and methods described herein can be used to with domesticated animals and/or pets.
  • the subject is human.
  • the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex.
  • adult and newborn subjects are intended to be covered.
  • subjects include humans, dogs, cats, cows, goats, and mice.
  • the term subject is further intended to include transgenic species.
  • the subject can be of European ancestry.
  • the subject can be of African American ancestry.
  • the subject can be of Asian ancestry.
  • parenteral administration refers to administration through injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
  • subcutaneous administration refers to administration just below the skin.
  • Intravenous administration means administration into a vein.
  • a dose refers to a specified quantity of a pharmaceutical agent provided in a single administration.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • a dosage unit refers to a form in which a pharmaceutical agent is provided.
  • a dosage unit is a vial comprising lyophilized antisense oligonucleotide.
  • a dosage unit is a vial comprising reconstituted antisense oligonucleotide.
  • Example 1 Aminooxy click chemistry (AOCC): a versatile bioconjugation approach [0777] Conjugation chemistry has invigorated oligonucleotide therapeutics field as demonstrated by the clinical success of siRNA conjugated to GalNAc, the ligand for the asialoglycoprotein (ASGPR) hepatocyte-specific receptor. Aminooxy-functionalized (-O-NH 2 ) sugars and nucleosides have attracted interest as they can be easily derivatized through oxime ligation (Rodriguez, E. C.; Marcaurelle, L. A.; Bertozzi, C. R. J. Org.
  • the inventors developed a simple “click type” amino-oxy-based chemistry that we call aminooxy click chemistry (AOCC) and used it to synthesize 2′-, 3′- and 5′-aminooxy nucleosides conjugated to bis-homo (I) and bis-hetero (II) ligands (FIG. 1)
  • the bis-homo ligand conjugation leads to bivalent ligand presentation.
  • the bis- hetero conjugation will allow placement of ligands with different chemical functions, for example, an aldehyde and an acid; and also ligands with different biological functions such as a targeting ligand and a pharmacokinetics modifier to the same -O-NH 2 linkage.
  • RNA interference-based therapeutics furthermore, we are in the process of testing whether these conjugates enhance better strand-bias for antisense strand incorporation into the RNA-induced silencing complex and improved metabolic stability from nucleases.
  • FR ⁇ Folate
  • integrins transmembrane receptors
  • aminooxy functionality which can form a new class of oligonucleotide drugs.
  • aminooxy group has been used in the oligo-nucleotide chemistry as an active group for conjugation of various group 2-6 as permanent or reversible functional group 7 , mostly the oxime formation has been utilized for such ligation. 6 When the oxime bond undergoes reductive amination, mostly the second alkyl groups used, were methyl (CH 3 ), ethyl, isopropyl etc.
  • aminooxy chemistry has been used widely in sugar 10-12 , aminooxy peptide 13-18 , and neutral nucleoside backbone chemistry 19-26 apart from conjugation chemistry which broadens its scope considerably.
  • the inventors have used aminooxy group in four different chemistries to demonstrate its application in the field of oligonucleotide synthesis and named it as aminooxy click chemistry (AOCC) approach.
  • the classifications are as follows: 1. Oxime type conjugation; 2. Mono substituted aminooxy (second ligand being simple CH 3 group); And more importantly 3. Bis-homo ligands conjugation. (same ligand, two times); and 4. Bis-hetero ligands conjugation. (different ligands, one after another).
  • Scheme 3 Synthesis of 2 ⁇ - and 3 ⁇ - modified amidites Ligand post-synthesis conjugation plan on solid support [0785] Monomers and 5 ⁇ -end modifications for AOCC on solid support are shown in Scheme 4 and 5.
  • Scheme 4. Synthesis of AOCC modified oligonucleotides from AOCC-conjugate building blocks and AOCC precursors:
  • Scheme 5 Schematic representation and amidites synthesized for AOCC on solid support
  • Scheme 6 Synthesis of exemplary 2 ⁇ - and 3 ⁇ -AOCC modified amidites and CPGs for conjugation at internal and 3 ⁇ -end conjugation of ONs on solid support are shown in Scheme 6.
  • Scheme 6 Synthesis of 2 ⁇ - and 3 ⁇ -modified amidites and CPGs for conjugation to ONs on the solid support
  • FIGS.3 Some exemplary 2 ⁇ - and 3 ⁇ - aminooxy conjugated nucleoside building blocks are shown in FIGS.3 (2 ⁇ -analogues) and 4 (3 ⁇ -analogues).
  • FIG. 5 shows some exemplary positions in nucleosides for AOCC: (1) Sugar modifications: 2 ⁇ (11b, 16, 17), 3 ⁇ (11a, 18.
  • FIG.6 depicts some exemplary monomers for AOCC at the nucleobase.
  • Exemplary non-nucleoside scaffolds for AOCC are shown in FIGS. 7 (Prolinol scaffolds), 8 (Serinol scaffolds), 9 (D- and L-Threoninol scaffolds), and 10 (conjugates derived from Pentaerythritol and Norbornyl scaffolds).
  • FIG. 12 shows single building block incorporation: (i) at 5 ⁇ -end of sense (red) / antisense (blue) strand and (ii) at 3 ⁇ -end of sense (red) / antisense (blue) strand. Note: antisense strand AOCC will be in the nucleobases and 2’ 3’ of sugars, not in the 5’ of sugar.
  • FIG. 13 shows double building block incorporation: (i) 5 ⁇ -end of sense (red) and 3 ⁇ - end of antisense (blue) strand; (ii) 3 ⁇ -end of sense (red) and 5 ⁇ -end of antisense (blue) strand; (iii) 3 ⁇ - and 5 ⁇ -end of sense (red) strand; (iv) 3 ⁇ - and 5 ⁇ -end of antisense (blue) strand.
  • antisense strand AOCC will be in the nucleobases and 2’ 3’ of sugars, not in the 5’ of sugar.
  • FIG. 14 shows incorporation of single building block at internal position: Single or multiple site modifications are possible either in sense or antisense strands.
  • the support was washed with pyridine, MeOH, ACN.
  • Oxime conjugation The support-bound oligonucleotides were treated with 1 mol/L aldehyde in DCM for 2 h at rt. The support was washed with DCM, MeOH, ACN.
  • Reductive amination The support-bound oligonucleotides were treated with freshly prepared 0.5 M solution of NaCNBH 3 in 75/25 DCM/MeOH with 1% AcOH as an additive for 2 h. The support was washed with DCM, MeOH, ACN.
  • ONs were cleaved from solid support and deprotected using ammonium hydroxide at 30 °C for 17 h. After filtration through a nylon syringe filter (0.45 ⁇ m), ONs were either stored for purification. ONs were purified using anion-exchange high-performance liquid chromatography (IEX-HPLC) using an appropriate gradient of mobile phase (0.15 M NaCl, 10% MeCN and 1.0 M NaBr, 10% MeCN) and desalted using size-exclusion chromatography with water as an eluent.
  • IEX-HPLC anion-exchange high-performance liquid chromatography
  • the oligonucleotide was manually released from support and deprotected using a mixture of aqueous MeNH 3 (40% wt) at 60 °C for 12 min or using 28-30% ammonium hydroxide solution at 60 °C for 5h. After filtration through a 0.45- ⁇ m nylon filter, oligonucleotides were either purified or, for oligonucleotides containing ribose sugars, the 2′ hydroxyl was deprotected by treatment with Et 3 N ⁇ 3HF at 60 °C for 30 min.
  • Oligonucleotides were purified using IEX-HPLC using an appropriate gradient of mobile phase (buffer A: 0.15 M NaCl, 10% CH 3 CN; buffer B 1.0 M NaBr, 10% MeCN) and desalted using size-exclusion chromatography with water as an eluent. Oligonucleotides were then quantified by measuring the absorbance at 260 nm. Extinction coefficients were calculated using the following extinction coefficients for each residue: A, 13.86; T/U, 7.92; C, 6.57; and G, 10.53 M -1 cm -1 . The purity and identity of modified ONs were verified by analytical anion exchange chromatography and mass spectrometry, respectively.
  • Precipitation of the crude oligonucleotides was accomplished via the addition of 1.2 mL of ACN/EtOH (9:1, v/v) to each well, followed by centrifugation at 3000 rpm for 45 min at 4 °C. The supernatant was removed from each well, and the pellets were resuspended in 950 ⁇ L of 20 mM aqueous NaOAc. Oligonucleotides were desalted over a GE Hi-Trap desalting column (Sephadex G25 Superfine) using water as an eluant. The identities and purities of all oligonucleotides were confirmed using ESI-MS and IEX-HPLC, respectively.
  • the 5 ⁇ -(S)-C-methyl-guanosine phosphoramidities were 0.15 M in anhydrous 15% DCM in CH 3 CN.
  • the oxidizing reagent was 0.02 M I2 in THF/pyridine/H 2 O.
  • the detritylation reagent was 3% dichloroacetic acid in CH 2 Cl 2 .
  • the support-bound oligonucleotides were treated with 0.5 mol/L hydrazine hydrate in Pyridine/AcOH (4:1 v/v) for 1 h at rt to deprotect phtalimido group.
  • the support was washed with pyridine, MeOH, ACN.
  • Oxime conjugation with (5Z,8Z,29Z,32Z)-heptatriaconta-5,8,29,32-tetraen-19-yl-4-((4,4- diethoxybutyl)amino)butan -oate (IV) was accomplished after treating the purified oligomer with 1 mol/L aldehyde in 20% DMF in water containing 2% acetic acid. The reaction mixture was kept at rt for 5 hrs and then dialyzed by 500 Dalton dialysis cut-off bag through overnight.
  • Aqueous solution was filtered through a nylon syringe filter (0.45 ⁇ m), ON was purified using anion- exchange high-performance liquid chromatography (IEX-HPLC) using an appropriate gradient of mobile phase (0.15 M NaCl, 10% MeCN and 1.0 M NaBr, 10% MeCN) and desalted using size- exclusion chromatography with water as an eluent. Collected fractions were lyophilized to afford the oligonucleotide.
  • IEX-HPLC anion- exchange high-performance liquid chromatography
  • exonuclease 150 mU/mL SVPDE or 500 mU/mL PDII was added immediately prior to analysis via IEX HPLC (dionex DNAPac PA200,4x250 mm) using a gradient of 37-52% mobile phase (1 M NaBr, 20 mM sodium phosphate, pH 11, 15% MeCN; stationary phase: 20 mM sodium phosphate, 15% MeCN, pH 11) over 7.5 min with a flow of 1 mL/min. Samples were analyzed at intervals for 24 h. Absorbance was monitored at 260 nm. The quantity of full-length ON was determined as the area under the curve.
  • the advantages of AOCC at 5 ⁇ -end of RNA strands include, but are not limited to the following: 1. Better strand bias - phosphorylation of sense strand will be blocked (RISC loading in vivo); 2. Potential Elimination of 2PS at the 5 ⁇ end of sense strand (improved nuclease resistance)- hindered ligand give exo-nuclease stability to sense strand (in vivo); 3. Multiple ligands conjugation; 4.
  • the reaction mixture was stirred at ambient temperature for 4 h under argon atmosphere.
  • the reaction mixture was diluted with CH 2 Cl 2 (100 mL) then washed with saturated aqueous NaHCO 3 .
  • the organic layer was separated, dried over anhydrous Na 2 SO 4 , filtered, and concentrated.
  • the reaction ⁇ ixture was stirred at ambient temperature for 5 h under argon atmosphere.
  • the reaction mixture was diluted with CH 2 Cl 2 (100 mL) then washed with saturated aqueous NaHCO 3 .
  • the organic layer was separated, dried over anhydrous Na 2 SO 4 , filtered, and concentrated.
  • the crude material was purified by flash silica gel column chromatography (50 – 80% EtOAc in hexanes) to give mono- and bis- benzoated compounds separately.
  • the reaction mixture was stirred at ambient temperature for 4 h under argon atmosphere.
  • the reaction mixture was diluted with CH 2 Cl 2 (100 mL) then washed with saturated aqueous NaHCO 3 .
  • the organic layer was separated, dried over anhydrous Na 2 SO 4 , filtered, and concentrated.
  • CPG ELN0132-514 [0859] Added ELN0132-472 (0.4 g, 434.80 ⁇ mol) and diisopropylethylamine (224.77 mg, 1.74 mmol, 302.93 ⁇ L) into rb flask. Then added dry acetonitrile (50 mL). Stirred well to dissolve and then HBTU (173.14 mg, 456.54 ⁇ mol) to preactivate acid, stirred for ⁇ 5 min, then added CPG. Capped and put on mechanical shaker overnight. Next day filtered CPG, washed with ACN, then MeOH, then ACN, then diethyl ether.
  • CPG ELN0132-515 [0866] Added ELN0132-473 (0.15 g, 163.05 ⁇ mol) and diisopropylethylamine (84.29 mg, 652.20 ⁇ mol, 113.60 ⁇ L) into rb flask. Then added dry acetonitrile (30 mL). Stirred well to dissolve and then HBTU (64.93 mg, 171.20 ⁇ mol) to preactivate acid. Let stirr for ⁇ 5 min, then added CPG. Capped and put on mechanical shaker overnight. Next day filtered CPG, washed with ACN, then MeOH, then ACN, then diethyl ether.
  • Scheme 17 Synthesis of amidite 15 and 16 [0869] Reductive amination of 10 with glutaraldehyde resulted in compound 17 with a six- membered heterocyclic ring comprising of the aminooxy nitrogen atom. Removal of TBS protecting group in presence of TBAF from 17 afforded compound 18. Phosphitylation reaction of 18 produced amidite 19 in moderate yield (Scheme 18).
  • Scheme 18 Synthesis of amidite 19 [0870] Reaction of 2-(2-oxoethoxy)acetaldehyde 32 with compound 10 under similar reductive amination condition, produced 20, the cyclic six-membered heterocyclic structure containing the aminooxy nitrogen atom at 5 ⁇ -position, however, in low yield.
  • citral (357.29 mg, 2.30 mmol, 402.35 ⁇ L) was added and stirred for 0.5 h and continued stirring for 30 minutes at 20 °C. Second portion of sodium cyanoborohydride (2.6 equiv) was then added to this reaction mixture and stirred for 2 hr. TLC showed completion of reaction. After diluting the reaction mixture with DCM (20 mL), water (20 mL) was added, and organic layer was separated. DCM layer dried over anhydrous CaCl 2 , filtered and filtrate was evaporated to dryness.
  • the reaction mixture was stirred for 1 hr at 15 °C and hexadecanal (141.91 mg, 590.25 ⁇ mol) in DCM (0.2 mL) was added. The stirring was continued for 30 min and additional amount of sodium cyanoborohydride (33.84 mg, 511.55 ⁇ mol) was added. The resulting mixture was stirred for another 2 hr and then diluted with DCM (10 mL) and washed with ice water. The organic layer was separated, dried over anhydrous Na 2 SO 4 and concentrated.
  • LNA-A analogues [0953] Methyl-16-[[(4R,6R)-6-(6-aminopurin-9-yl)-7-[tert-butyl(dimethyl)silyl]oxy-2,5- dioxabicyclo[2.2.1]heptan-4-yl]methoxy-hexyl-amino]hexadecanoate ELN0132-272: [0954] To a solution of O-[[(4R,6R)-6-(6-aminopurin-9-yl)-7-[tert-butyl(dimethyl)silyl]oxy- 2,5-dioxabicyclo[2.2.1]heptan-4-yl]methyl]hydroxylamine (0.7 g, 1.71 mmol) in DCM (13.87 mL)was added Hexanal (262.69 mg, 2.57 mmol, 314.97 ⁇ L, 98% purity) at 22 °C.
  • Reaction mixture was stirred for 1 hr and methyl 16-oxohexadecanoate (731.05 mg, 2.57 mmol, 501.94 ⁇ L) was added into it. Stirring continude for 1 hr and then second batch of sodium cyanoborohydride (294.69 mg, 4.46 mmol, 95% purity) was added. Reaction mixture was diluted with DCM (20 mL) after 3 hrs. Orgainc layer was washed with water (10 mL), brine (2 x 20 mL) and organic layer was separated. DCM layer was dried over anhydrous Na 2 SO 4 , filtered and the filtrate was evaporated to dryness.
  • N-(((2R,3R,4R,5R)-3-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4- dihydropyrimidin-1(2H)-yl)-4-methoxytetrahydrofuran-2-yl)methoxy)palmitamide (57, ELN0132-16L): To a solution of palmitic acid (160.42 mg, 619.35 umol, 188.07 uL) in dimethylformamide (3 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (118.73 mg, 619.35 umol) and DIPEA (134.76 mg, 1.03 mmol, 181.61 uL) were added and stirred for 10 minutes at 25 °C.

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