WO2017132669A1 - Branched oligonucleotides - Google Patents

Branched oligonucleotides Download PDF

Info

Publication number
WO2017132669A1
WO2017132669A1 PCT/US2017/015633 US2017015633W WO2017132669A1 WO 2017132669 A1 WO2017132669 A1 WO 2017132669A1 US 2017015633 W US2017015633 W US 2017015633W WO 2017132669 A1 WO2017132669 A1 WO 2017132669A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
independently
formula
occurrence
nucleotides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2017/015633
Other languages
English (en)
French (fr)
Inventor
Anastasia Khvorova
Matthew Hassler
Julia ALTERMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Massachusetts Amherst
Original Assignee
University of Massachusetts Amherst
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Massachusetts Amherst filed Critical University of Massachusetts Amherst
Priority to EP17745083.0A priority Critical patent/EP3408391A4/en
Priority to CA3011894A priority patent/CA3011894A1/en
Priority to AU2017210726A priority patent/AU2017210726B2/en
Priority to HK19101058.6A priority patent/HK1258758A1/zh
Priority to JP2018539903A priority patent/JP7749201B6/ja
Publication of WO2017132669A1 publication Critical patent/WO2017132669A1/en
Anticipated expiration legal-status Critical
Priority to JP2022200533A priority patent/JP2023040012A/ja
Priority to AU2023206109A priority patent/AU2023206109A1/en
Priority to JP2024232308A priority patent/JP2025066730A/ja
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/08—Deoxysugars; Unsaturated sugars; Osones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H99/00—Subject matter not provided for in other groups of this subclass
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/11—Antisense
    • 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
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/52—Physical structure branched

Definitions

  • This disclosure relates to novel branched oligonucleotides designed to achieve unexpectedly high efficacy, uptake and tissue distribution.
  • RNA interference RNA interference
  • siRNA siRNA
  • therapeutic oligonucleotides are simple and effective tools for a variety of applications, including the inhibition of gene function.
  • An example of such inhibition is RNA interference (RNAi).
  • RNAi RNA interference
  • the present disclosure provides branched oligonucleotides ("compounds of the invention”) exhibiting unexpected improvement in distribution, in vivo efficacy and safety.
  • a branched oligonucleotide compound comprising two or more nucleic acids, the nucleic acids are connected to one another by one or more moieties selected from a linker, a spacer and a branching point.
  • the branched oligonucleotide comprises 2, 3, 4, 6 or 8 nucleic acids.
  • each nucleic acid is single- stranded and has a 5' end and a 3 ' end, and each nucleic acid is independently connected to a linker, a spacer, or a branching point at the 5 ' end or at the 3 ' end.
  • each single-stranded nucleic acid independently comprises at least 15 contiguous nucleotides.
  • the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides, and has complementarity to a target.
  • each nucleic acid comprises one or more chemically-modified nucleotides. In an embodiment, each nucleic acid consists of chemically-modified nucleotides.
  • each nucleic acid is double- stranded and comprises a sense strand and an antisense strand, the sense strand and the antisense strand each have a 5 ' end and a 3 ' end.
  • each double-stranded nucleic acid is independently connected to a linker, spacer or branching point at the 3 ' end or at the 5 ' end of the sense strand or the antisense strand.
  • the sense strand and the antisense strand each comprise one or more chemically-modified nucleotides. In an embodiment, the sense strand and the antisense strand each consist of chemically-modified nucleotides. In an embodiment, the sense strand and the antisense strand both comprise alternating 2'-methoxy-nucleotides and 2'-fluoro- nucleotides. In an embodiment, the nucleotides at positions 1 and 2 from the 5 ' end of the sense and antisense strands are connected to adjacent nucleotides via phosphorothioate linkages. In an embodiment, the nucleotides at positions 1 -6 from the 3 ' end, or positions 1 -7 from the 3 ' end, are connected to adjacent nucleotides via phosphorothioate linkages.
  • the branched oligonucleotide further comprises a hydrophobic moiety.
  • the hydrophobic moiety is attached to one or more terminal 5' positions of the branched oligonucleotide compound.
  • the hydrophobic moiety may be comprised within one or more 5 ' phosphate moieties.
  • the hydrophobic moiety comprises an alkyl or alkenyl moiety (e.g. , an alkyl or alkenyl chain, or a saturated or unsaturated fatty acid residue), a vitamin or cholesterol derivative, an aromatic moiety (e.g., phenyl or naphthyl), a lipophilic amino acid or a combination thereof.
  • each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent.
  • L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and
  • formula (I) optionally further comprises one or more branch point B, and one or more spacer S;
  • B is independently for each occurrence a polyvalent organic species or derivative thereof;
  • S is independently for each occurrence selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof;
  • N is an RNA duplex comprising a sense strand and an antisense strand, the sense strand and antisense strand each independently comprise one or more chemical modifications; and
  • n is 2, 3, 4, 5, 6, 7 or 8.
  • the compound of formula (I) has a structure selected from formulas (I-l)-(I-9) of Table 1.
  • each antisense strand independently comprises a 5 ' terminal group R selected from the groups of Table 2.
  • X for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof
  • Y for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof
  • - represents a phosphodiester internucleoside linkage
  • represents a phosphorothioate internucleoside linkage
  • — represents, individually for each occurrence, a base-pairing interaction or a mismatch.
  • the compound of formula (I) has the structure of formula (III):
  • X for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'-fluoro modification
  • X for each occurrence, independently, is a nucleotide comprising a 2'-0- methyl modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'- deoxy-2'-fluoro modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-0-methyl modification.
  • the compound of formula (I) has the structure of formula (IV):
  • A is an adenosine comprising a 2'-deoxy-2'-fluoro modification
  • A is an adenosine comprising a 2'-0-methyl modification
  • G is an guanosine comprising a 2'-deoxy-2'-fluoro modification
  • G is an guanosine comprising a 2'-0-methyl modification
  • U is an uridine comprising a 2'-deoxy-2'-fluoro modification
  • U is an uridine comprising a 2'-0-methyl modification
  • C is an cytidine comprising a 2'-deoxy-2'-fluoro modification
  • C is an cytidine comprising a 2'-0-methyl modification.
  • the compound of formula (I) has the structure of formula (V):
  • (V) X for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof;
  • Y for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof;
  • - represents a phosphodiester internucleoside linkage;
  • represents a phosphorothioate internucleoside linkage;
  • — represents, individually for each occurrence, a base-pairing interaction or a mismatch.
  • the compound of formula (I) has the structure of formula (VI):
  • X for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'-fluoro modification
  • X for each occurrence, independently, is a nucleotide comprising a 2'-0- methyl modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'- deoxy-2'-fluoro modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-0-methyl modification.
  • the compound of formula (I) has the structure of formula (VII):
  • A is an adenosine comprising a 2'-deoxy-2'-fluoro modification
  • A is an adenosine comprising a 2'-0-methyl modification
  • G is an guanosine comprising a 2'-deoxy-2'-fluoro modification
  • G is an guanosine comprising a 2'-0-methyl modification
  • U is an uridine comprising a 2'-deoxy-2'-fluoro modification
  • U is an uridine comprising a 2'-0-methyl modification
  • C is an cytidine comprising a 2'-deoxy-2'-fluoro modification
  • C is an cytidine comprising a 2'-0-methyl modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'-fluoro modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-0-methyl modification.
  • R is R 3 and n is 2.
  • L has the structure of L2:
  • R is R 3 and n is 2.
  • a delivery system for therapeutic nucleic acids having the structure of formula (VIII):
  • L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, formula (VIII) optionally further comprises one or more branch point B, and one or more spacer S; B is independently for each occurrence a polyvalent organic species or derivative thereof; S is independently for each occurrence selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; each cNA, independently, is a carrier nucleic acid comprising one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8.
  • the compound of formula (VIII) has a structure selected from formulas (VIII-l)-(VIII-9) of Table 3: Table 3
  • each cNA independently comprises at least 15 contiguous nucleotides. In an embodiment, each cNA independently consists of chemically-modified nucleotides.
  • the delivery system further comprises n therapeutic nucleic acids (NA), each NA is hybridized to at least one cNA.
  • NA therapeutic nucleic acids
  • each NA independently comprises at least 16 contiguous nucleotides. In an embodiment, each NA independently comprises 16-20 contiguous nucleotides. In an embodiment, each NA comprises an unpaired overhang of at least 2 nucleotides. In an embodiment, the nucleotides of the overhang are connected via phosphorothioate linkages.
  • each NA independently, is selected from the group consisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, or guide RNAs. In an embodiment, each NA is the same. In an embodiment, each NA is not the same.
  • the delivery system further comprising n therapeutic nucleic acids (NA) has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein.
  • the target of delivery is selected from the group consisting of: brain, liver, skin, kidney, spleen, pancreas, colon, fat, lung, muscle, and thymus.
  • FIG. 1 shows the structure of Di-hsiRNAs. Black - 2'-0-methyl, grey - 2'-fluoro, red dash - phosphorothioate bond, linker - tetraethylene glycol. Di-hsiRNAs are two asymmetric siRNAs attached through the linker at the 3 ' ends of the sense strand. Hybridization to the longer antisense strand creates protruding single stranded fully phosphorthioated regions, essential for tissue distribution, cellular uptake and efficacy. The structures presented utilize teg linger of four monomers. The chemical identity of the linker can be modified without the impact on efficacy. It can be adjusted by length, chemical composition (fully carbon), saturation or the addition of chemical targeting ligands.
  • FIG. 2 shows a chemical synthesis, purification and QC of Di-branched siRNAs.
  • FIG. 3 shows HPLC and QC of compounds produced by the method depicted in Figure 2.
  • Three major products were identified by mass spectrometry as sense strand with TEG (tetraethylene glycol) linker, di-branched oligo and Vit-D (calciferol) conjugate. All products where purified by HPLC and tested in vivo independently. Only Di branched oligo is characterized by unprecedented tissue distribution and efficacy, indicating that branching structure is essential for tissue retention and distribution.
  • FIG. 4 shows mass spectrometry confirming the mass of the Di-branched oligonucleotide.
  • the observed mass of 11683 corresponds to two sense strands attached through the TEG linker by the 3 ' ends.
  • FIG. 5 shows a synthesis of a branched oligonucleotide using alternative chemical routes.
  • FIG. 6 shows exemplary amidite linkers, spacers and branching moieties.
  • FIG. 7 shows oligonucleotide branching motifs.
  • the double-helices represented oligonucleotides.
  • the combination of different linkers, spacer and branching points allows generation of a wide diversity of branched hsiRNA structures.
  • FIG. 8 shows structurally diverse branched oligonucleotides.
  • FIG. 9 shows an asymmetric compound of the invention having four single-stranded phosphorothioate regions.
  • FIG. 10 shows in vitro efficacy data.
  • A HeLa cells were transfected (using RNAiMax) with Di-branched oligo at concentrations shown for 72 hours.
  • B Primary cortical mouse neurons were treated with Di-branched oligo at concentrations shown for 1 week. mRNA was measured using Affymetrix Quantigene 2.0. Data was normalized to housekeeping gene (PPIB) and graphed as % of untreated control.
  • PPIB housekeeping gene
  • C HeLa cells were treated passively (no formulation) with Di-siRNA oligo at concentrations shown for 1 week.
  • FIG. 11 shows brain distribution of Di-siRNA or TEG only after 48 hours following intra-striatal injection.
  • N 2 mice per conjugate.
  • the left side of brain in (A) appears bright red, whereas the left side of the brain in (B) only faintly red.
  • FIG. 12 shows that a single injection of Di-siRNA was detected both ipsilateral and contralateral to the injection site.
  • FIG. 13 shows Di-hsiRNA wide distribution and efficacy in mouse brain.
  • A Robust Htt mRNA silencing in both Cortex and Striatum 7 days after single IS injection (25 ug), QuantiGene®.
  • B Levels of hsiRNA accumulation in tissues 7 days after injection (PNA assay).
  • FIG. 14 shows wide distribution and efficacy throughout the spinal cord following bolus intrathecal injection of Di-hsiRNA. Intrathecal injection in lumbar of 3 nmols Di- branched Oligo (6 nmols of corresponding antisense HTT strand).
  • FIG. 15 shows branched oligonucleotides of the invention, (A) formed by annealing three oligonucleotides.
  • the longer linking oligonucleotides may comprise a cleavable region in the form of unmodified RNA, DNA or UNA;
  • C branched oligonucleotides made up of three separate strands.
  • the long dual sense strand can be synthesized with 3' phosphoramidites and 5' phosphoramidites to allow for 3 '-3' adjacent or 5 '-5 ' adjacent ends.
  • FIG. 16 shows branched oligonucleotides of the invention with conjugated bioactive moieties.
  • FIG. 17 shows the relationship between phosphorothioate content and
  • FIG. 18 depicts exemplary hydrophobic moieties.
  • FIG. 19 depicts exemplary internucleotide linkages.
  • FIG. 20 depicts exemplary internucleotide backbone linkages.
  • FIG. 21 depicts exemplary sugar modifications.
  • FIG. 22 depicts Di-FM-hsiRNA.
  • A Chemical composition of the four sub-products created from VitD-FM-hsiRNA synthesis and crude reverse phase analytical HPLC of the original chemical synthesis.
  • B Efficacy of sub-products in HeLa cells after lipid mediated delivery of hsiRN A. Cells were treated for 72 hours. mRNA was measured using
  • QuantiGene 2.0 kit (Affymetrix). Data are normalized to housekeeping gene HPRT and presented as a percent of untreated control.
  • C A single, unilateral intrastriatal injection (25 ⁇ g) of each hsiRNA sub-product. Images taken 48 hours after injection.
  • FIGS. 23A-B show that Di-HTT-Cy3does not effectively induce silencing in the liver or kidneys following intrastriatal injection.
  • FIG. 23A depicts a scatter dot plot showing Htt mRNA expression in the liver one week post intrastriatal injection of Di-HTT-Cy3 compared to a negative control (aCSF).
  • FIG. 23B depicts a scatter dot plot showing Htt mRNA expression in the kidney one week post intrastriatal injection of Di-HTT-Cy3 compared to a negative control (aCSF).
  • FIGS. 23A-B show that Di-HTT-Cy3does not effectively induce silencing in the liver or kidneys following intrastriatal injection.
  • FIG. 23A depicts a scatter dot plot showing Htt mRNA expression in the liver one week post intrastriatal injection of Di-HTT-Cy3 compared to a negative control (aCSF).
  • aCSF negative control
  • FIG. 24A-B show that Di-HTT effectively silences HTT gene expression in both the striatum and the cortex following intrastriatal injection and that Di-HTT-Cy3 is slightly more efficacious than Di-HTT (unlabeled).
  • FIG. 24A depicts a scatter dot plot showing Htt mRNA expression in the striatum one week post intrastriatal injection of Di-HTT, Di-HTT-Cy3, or two negative controls (aCSF or Di-NTC).
  • FIG. 24B depicts a scatter dot plot showing Htt mRNA expression in the cortex one week post intrastriatal injection of Di-HTT, Di-HTT- Cy3, or two negative controls (aCSF or Di-NTC).
  • FIG. 25 depicts a scatter dot plot measuring Di-HTT-Cy3 levels in the striatum and cortex. The plot shows that significant levels of Di-HTT-Cy3 are still detectable two weeks post intrastriatal injection.
  • FIGS. 26A-B show that Di-HTT-Cy3 effectively silences HTT mRNA and protein expression in both the striatum and the cortex two weeks post intrastriatal injection.
  • FIG. 26A depicts a scatter dot plot measuring Htt mRNA levels in the striatum and cortex two weeks post injection.
  • FIG. 26B depicts a scatter dot plot measuring Htt protein levels in the striatum and cortex two weeks post injection.
  • FIGS. 27A-B show that high dose Di-HTT-Cy3 treatment does not cause significant toxicity in vivo but does lead to significant gliosis in vivo two weeks post intrastriatal injection.
  • FIG. 27A depicts a scatter dot plot measuring DARPP32 signal in the striatum and cortex two weeks after injection with Di-HTT-Cy3 or aCSF.
  • FIG. 27B depicts a scatter dot plot measuring GFAP protein levels in the striatum and cortex two weeks after injection with Di-HTT-Cy3 or aCSF.
  • FIG. 28 depicts fluorescent imaging showing that intrathecal injection of Di-HTT-Cy3 results in robust and even distribution throughout the spinal cord.
  • FIG. 29 depicts a merged fluorescent image of FIG. 28B (zoom of spinal cord). Blue- nuclei, red-Di-HTT-Cy3.XXX
  • FIGS. 30A-C shows the widespread distribution of Di-HTT-Cy3 48 hours post intracerebro ventricular injection.
  • FIG. 30A depicts fluorescent imaging of sections of the striatum, cortex, and cerebellum.
  • FIG. 30B depicts brightfield images of the whole brain injected with control (aCSF) or Di-HTT-Cy3.
  • FIG. 30C depicts a fluorescent image of a whole brain section 48 hours after Di-HTT-Cy3 injection.
  • FIG. 31 shows that Di-HTT-Cy3 accumulates in multiple brain regions two weeks post intracerebro ventricular injection. A scatter dot plot measures the level of Di-HTT-Cy3 in multiple areas of the brain.
  • FIG. 32A shows that Di-HTT-Cy3 induces Htt gene silencing in multiple regions of the brain two weeks post intracerebroventricular injection compared to a negative control injection (aCSF).
  • a scatter dot plot measures Htt mRNA levels in multiple areas of the brain.
  • FIG. 32B shows that Di-HTT-Cy3 induces Htt silencing in multiple regions of the brain two weeks post intracerebroventricular injection compared to a negative control injection (aCSF).
  • a scatter dot plot measures Htt protein levels in multiple areas of the brain.
  • FIG. 33 shows that intracerebroventricular injection of high dose Di-HTT-Cy3 causes minor toxicity in vivo.
  • a scatter dot plot measures DARPP32 signal in multiple regions of the brain following Di-HTT-Cy3 of aCSF injection.
  • FIG. 34 shows that intracerebroventricular injection of high dose Di-HTT-Cy3 causes significant gliosis in vivo.
  • a scatter dot plot measures DARPP32 signal in multiple regions of the brain following Di-HTT-Cy3 of aCSF injection.
  • FIG. 35 shows that Di-HTT-Cy3 is distributed to multiple organs following intravenous injection. Fluorescent images depict Di-HTT-Cy3 levels in the heart, kidney, adrenal gland, and spleen following intravenous injection of Di-HTT-Cy3 or a negative control (PBS).
  • PBS negative control
  • FIG. 36 shows that Di-HTT-Cy3 accumulates in multiple organs following intravenous injection.
  • a scatter dot plot measures the levels of Di-HTT-Cy3 in multiple tissues.
  • FIG. 37 illustrates the structures of hsiRNA and fully metabolized (FM) hsiRNA.
  • FIGS. 38A-B show that full metabolic stabilization of hsiRNAs results in more
  • FIG. 38A depicts a scatter dot plot measuring HTT mRNA levels up to 12 days after intrastriatal injection.
  • FIG. 38B depicts a scatter dot plot measuring HTT mRNA levels up to 28 days after intrastriatal injection.
  • FIG. 39 depicts the chemical diversity of single stranded fully modified
  • the single stranded oligonucleotides can consist of gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, or PNAs.
  • FIG. 40 depicts Di-HTT with a TEG phosphoramidate linker.
  • FIG. 41 depicts Di-HTT with a TEG di-phosphate linker.
  • FIG. 42 depicts variations of Di-HTT with either two oligonucleotide branches or four oligonucleotide branches.
  • FIG. 43 depicts another variant of Di-HTT of a structure with two oligonucleotide branches and R2 attached to the linker.
  • FIG. 44 depicts a first strategy for the incorporation of a hydrophobic moeity into the branched oligonucleotide structures.
  • FIG. 45 depicts a second strategy for the incorporation of a hydrophobic moeity into the branched oligonucleotide structures.
  • FIG. 46 depicts a third strategy for the incorporation of a hydrophobic moeity into the branched oligonucleotide structures.
  • the present disclosure provides branched oligonucleotides ("compounds of the invention”) exhibiting unexpected improvement in distribution, in vivo efficacy and safety.
  • the branched oligonucleotides described herein efficiently and stably delivered small RNAs to multiple regions of the brain and multiple other organs, demonstrating unprecedented efficacy of delivery that has not been previously demonstrated with unconjugated small RNAs.
  • compositions described herein allow efficient, stable delivery of siRNA in order to promote potent silencing of therapeutic target genes.
  • the compositions exhibit therapeutic potential for many hard to treat diseases and overcome present challenges in employing RNA therapeutics.
  • a branched oligonucleotide compound comprising two or more nucleic acids, wherein the nucleic acids are connected to one another by one or more moieties selected from a linker, a spacer and a branching point.
  • branched oligonucleotides referred to as compounds of the invention.
  • compounds of the invention have two to eight oligonucleotides attached through a linker.
  • the linker may be hydrophobic.
  • compounds of the invention have two to three oligonucleotides.
  • the oligonucleotides independently have substantial chemical stabilization (e.g., at least 40% of the constituent bases are chemically-modified).
  • the oliogonucleotides have full chemical stabilization (i.e., all of the constituent bases are chemically-modified).
  • compounds of the invention comprise one or more single-stranded phosphorothioated tails, each independently having two to twenty nucleotides.
  • each single-stranded tail has eight to ten nucleotides.
  • compounds of the invention are characterized by three properties: (1 ) a branched structure, (2) full metabolic stabilization, and (3) the presence of a single-stranded tail comprising phosphorothioate linkers.
  • compoudns of the invention have 2 or 3 branches. The increased overall size of the branched structures promote increased uptake. Also, without being bound by a particular theory of activity, multiple adjacent branches (e.g. , 2 or 3) allow each branch to act cooperatively and thus dramatically enhance rates of internalization, trafficking and release.
  • compounds of the invention are characterized by the following properties: (1) two or more branched oligonucleotides, e.g. , wherein there is a non- equal number of 3' and 5 ' ends; (2) substantially chemically stabilized, e.g., wherein more than 40%, optimally 100%, of oligonucleotides are chemically modified (e.g. , no RNA and optionally no DNA); and (3) phoshorothioated single oligonucleotides containing at least 3, optimally 5-20 phosphorothioated bonds.
  • oligonucleotides attached at the branching points are single stranded and consist of miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs. These single strands can be attached at their 3 ' or 5 ' end. Combinations of siRNA and single stranded oligonucleotides could also be used for dual function. In another embodiment, short oligonucleotides complementary to the gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used to carry these active single-stranded oligonucleotides and enhance distribution and cellular internalization.
  • Di-siRNA compounds of the invention may comprise chemically diverse conjugates. Conjugated bioactive ligands may be used to enhance cellular specificity and to promote membrane association, internalization, and serum protein binding. Examples of bioactive moieties to be used for conjugation include DHAg2, DHA, GalNAc, and cholesterol. These moieties can be attached to Di-siRNA either through the connecting linker or spacer, or added via an additional linker or spacer attached to another free siRNA end.
  • Compounds of the invention have unexpectedly uniform distribution throughout the spinal cord and brain. Moreover, compounds of the invention exhibit unexpectedly efficient systemic delivery to a variety of tissues, and very high levels of tissue accumulation.
  • Compounds of the invention comprise a variety of therapeutic oligonucleotides, including including ASOs, miRNAs, miRNA inhibitors, splice switching, PMOs, PNAs.
  • compounds of the invention further comprise conjugated hydrophobic moieties and exhibit unprecedented silencing and efficacy in vitro and in vivo.
  • Non-limiting embodiments of branched oligonucleotide configurations are disclosed in Figures 1 , 7-9, 15-17, and 40-45.
  • Non-limiting examples of linkers, spacers and branching points are disclosed in Figure 6.
  • the branched oligonucleotide comprises 2, 3, 4, 6 or 8 nucleic acids. In one embodiment, the branched oligonucleotide comprises 2 nucleic acids. In another embodiment, the branched oligonucleotide comprises 3 nucleic acids. In another embodiment, the branched oligonucleotide comprises 4 nucleic acids. In another embodiment, the branched oligonucleotide comprises 6 nucleic acids. In another embodiment, the branched oligonucleotide comprises 8 nucleic acids. In another embodiment, the branched oligonucleotide comprises 5 nucleic acids. In another embodiment, the branched oligonucleotide comprises 7 nucleic acids.
  • each nucleic acid is single- stranded and has a 5' end and a 3 ' end, and each nucleic acid is independently connected to a linker, a spacer, or a branching point at the 5 ' end or at the 3' end.
  • each nucleic acid is connected to a linker, spacer or branching point at the 3' end.
  • each nucleic acid is connected to a linker, spacer or branching point at the 5' end.
  • each nucleic acid is connected to a linker.
  • each nucleic acid is connected to a spacer.
  • each nucleic acid is connected to a branch point.
  • each single-stranded nucleic acid independently comprises at least 15 contiguous nucleotides.
  • the nucleic acid comprises at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides, and has complementarity to a target.
  • the complementarity is >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%.
  • the nucleic acid has perfect complementarity to a target.
  • each nucleic acid is double- stranded and comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand each have a 5' end and a 3' end.
  • each double- stranded nucleic acid is independently connected to a linker, spacer or branching point at the 3' end or at the 5' end of the sense strand or the antisense strand.
  • each nucleic acid is connected to a linker, spacer or branching point at the 3 ' end of the sense strand.
  • each nucleic acid is connected to a linker, spacer or branching point at the 3' end of the antisense strand. In another embodiment, each nucleic acid is connected to a linker, spacer or branching point at the 5' end of the sense strand. In another embodiment, each nucleic acid is connected to a linker, spacer or branching point at the 5' end of the antisense strand. In one embodiment, each nucleic acid is connected to a linker. In another embodiment, each nucleic acid is connected to a spacer. In another embodiment, each nucleic acid is connected to a branch point.
  • each double-stranded nucleic acid independently comprises at least 15 contiguous nucleotides.
  • the antisense strand comprises at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 contiguous nucleotides, and has complementarity to a target.
  • the complementarity is >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50%.
  • the antisense strand has perfect complementarity to a target.
  • each nucleic acid comprises one or more chemically-modified nucleotides. In an embodiment, each nucleic acid consists of chemically-modified nucleotides. In certain embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of each nucleic acid comprises chemically-modified nucleotides.
  • the sense strand and the antisense strand each comprise one or more chemically-modified nucleotides.
  • the sense strand and the antisense strand each consist of chemically-modified nucleotides.
  • the sense strand and the antisense strand both comprise alternating 2'-methoxy-nucleotides and 2'-fluoro- nucleotides.
  • the nucleotides at positions 1 and 2 from the 5 ' end of the sense and antisense strands are connected to adjacent nucleotides via phosphorothioate linkages.
  • the nucleotides at positions 1 -6 from the 3 ' end, or positions 1 -7 from the 3 ' end are connected to adjacent nucleotides via phosphorothioate linkages. In other embodiments, at least 5 nucleotides are connected to adjacent nucleotides via phosphorothioate linkages.
  • each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent.
  • each linker is an ethylene glycol chain.
  • each linker is an alkyl chain.
  • each linker is a peptide.
  • each linker is RNA.
  • each linker is DNA. In another embodiment, each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is a phosphoramidate. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is a triazole. In another embodiment, each linker is a structure selected from the formulas of Figure 7.
  • L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and
  • formula (I) optionally further comprises one or more branch point B, and one or more spacer S; wherein B is independently for each occurrence a polyvalent organic species or derivative thereof; S is independently for each occurrence selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof;
  • N is an RNA duplex comprising a sense strand and an antisense strand, wherein the sense strand and antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8.
  • the compound of formula (I) has a structure selected from formulas (I-l)-(I-9) of Table 1.
  • the compound of formula (I) is formula (I-l ). In another embodiment, the compound of formula (I) is formula (1-2). In another embodiment, the compound of formula (I) is formula (1-3). In another embodiment, the compound of formula (I) is formula (1-4). In another embodiment, the compound of formula (I) is formula (1-5). In another embodiment, the compound of formula (I) is formula (1-6). In another embodiment, the compound of formula (I) is formula (1-7). In another embodiment, the compound of formula (I) is formula (1-8). In another embodiment, the compound of formula (I) is formula (1-9).
  • each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent.
  • each linker is an ethylene glycol chain.
  • each linker is an alkyl chain.
  • each linker is a peptide.
  • each linker is RNA. In another embodiment of the compound of formula (I), each linker is DNA. In another embodiment of the compound of formula (I), each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment of the compound of formula (I), each linker is a phosphoramidate. In another embodiment of the compound of formula (I), each linker is an ester. In another embodiment of the compound of formula (I), each linker is an amide. In another embodiment of the compound of formula (I), each linker is a triazole. In another embodiment of the compound of formula (I), each linker is a structure selected from the formulas of Figure 7.
  • B is a polyvalent organic species. In another embodiment of the compound of formula (I), B is a derivative of a polyvalent organic species. In one embodiment of the compound of formula (I), B is a triol or tetrol derivative. In another embodiment, B is a tri- or tetra-carboxylic acid derivative. In another embodiment, B is an amine derivative. In another embodiment, B is a tri- or tetra-amine derivative. In another embodiment, B is an amino acid derivative. In another embodiment of the compound of formula (I), B is selected from the formulas of Figure 6.
  • Polyvalent organic species are moieties comprising carbon and three or more valencies (i. e., points of attachment with moieties such as S, L or N, as defined above).
  • Non- limiting examples of polyvalent organic species include triols (e.g.
  • tetrols e.g., ribose, pentaerythritol, 1 ,2,3,5-tetrahydroxybenzene, and the like
  • tri-carboxylic acids e.g., citric acid, 1 ,3,5-cyclohexanetricarboxylic acid, trimesic acid, and the like
  • tetra-carboxylic acids e.g. , ethyl enediaminetetraacetic acid, pyromellitic acid, and the like
  • tertiary amines e.g.
  • tripropargylamine triethanolamine, and the like
  • triamines e.g. , diethyl enetriamine and the like
  • tetramines and species comprising a combination of hydroxyl, thiol, amino, and/or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, and the like).
  • each nucleic acid comprises one or more chemically-modified nucleotides. In an embodiment of the compound of formula (I), each nucleic acid consists of chemically-modified nucleotides. In certain embodiments of the compound of formula (I), >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of each nucleic acid comprises chemically-modified nucleotides.
  • each antisense strand independently comprises a 5 ' terminal group R selected from the groups of Table 2.
  • R is Ri. In another embodiment, R is R2. In another embodiment, R is R3. In another embodiment, R is R4. In another embodiment, R is R5. In another embodiment, R is R6. In another embodiment, R is R7. In another embodiment, R is Rs.
  • X for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof
  • Y for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically- modified derivatives thereof
  • - represents a phosphodiester internucleoside linkage
  • represents a phosphorothioate internucleoside linkage
  • — represents, individually for each occurrence, a base-pairing interaction or a mismatch.
  • the structure of formula (II) does not contain mismatches. In one embodiment, the structure of formula (II) contains 1 mismatch. In another embodiment, the compound of formula (II) contains 2 mismatches. In another embodiment, the compound of formula (II) contains 3 mismatches. In another embodiment, the compound of formula (II) contains 4 mismatches. In an embodiment, each nucleic acid consists of chemically-modified nucleotides.
  • >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of X's of the structure of formula (II) are chemically-modified nucleotides.
  • >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50 of X's of the structure of formula (II) are chemically-modified nucleotides.
  • the compound of formula (I) has the structure of formula (III):
  • X for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'- fluoro modification
  • X for each occurrence, independently, is a nucleotide comprising a 2'- O-methyl modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'-fluoro modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2 '-O-methyl modification.
  • X is chosen from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, X is chosen from the group consisting of 2'-0-methyl modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2'-0-methyl modified adenosine, guanosine, uridine or cytidine.
  • the structure of formula (III) does not contain mismatches. In one embodiment, the structure of formula (III) contains 1 mismatch. In another embodiment, the compound of formula (III) contains 2 mismatches. In another embodiment, the compound of formula (III) contains 3 mismatches. In another embodiment, the compound of formula (III) contains 4 mismatches.
  • the compound of formula (I) has the structure of formula (IV):
  • A is an adenosine comprising a 2'-deoxy-2'-fiuoro modification
  • A is an adenosine comprising a 2'-0-methyl modification
  • G is an guanosine comprising a 2'-deoxy-2'-fluoro modification
  • G is an guanosine comprising a 2'-0-methyl modification
  • U is an uridine comprising a 2'-deoxy-2'-fiuoro modification
  • U is an uridine comprising a 2'-0-methyl modification
  • C is an cytidine comprising a 2'-deoxy-2'-fiuoro modification
  • C is an cytidine comprising a 2'-0-methyl modification.
  • the compound of formula (I) has the structure of formula (V):
  • X for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof
  • Y for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically- modified derivatives thereof
  • - represents a phosphodiester internucleoside linkage
  • represents a phosphorothioate internucleoside linkage
  • — represents, individually for each occurrence, a base-pairing interaction or a mismatch.
  • the structure of formula (V) does not contain mismatches. In one embodiment, the structure of formula (V) contains 1 mismatch. In another embodiment, the compound of formula (V) contains 2 mismatches. In another embodiment, the compound of formula (V) contains 3 mismatches. In another embodiment, the compound of formula (V) contains 4 mismatches. In an embodiment, each nucleic acid consists of chemically-modified nucleotides.
  • >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of X's of the structure of formula (II) are chemically-modified nucleotides. In other embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of X's of the structure of formula (II) are chemically-modified nucleotides.
  • the compound of formula (I) has the structure of formula (VI): 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20
  • X for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'- fluoro modification
  • X for each occurrence, independently, is a nucleotide comprising a 2'- O-methyl modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'-fluoro modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-0-methyl modification.
  • X is chosen from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, X is chosen from the group consisting of 2'-0-methyl modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2'-0-methyl modified adenosine, guanosine, uridine or cytidine.
  • the structure of formula (VI) does not contain mismatches. In one embodiment, the structure of formula (VI) contains 1 mismatch. In another embodiment, the compound of formula (VI) contains 2 mismatches. In another embodiment, the compound of formula (VI) contains 3 mismatches. In another embodiment, the compound of formula (VI) contains 4 mismatches.
  • the compound of formula (I) has the structure of formula (VII):
  • A is an adenosine comprising a 2'-deoxy-2'-fluoro modification
  • A is an adenosine comprising a 2'-0-methyl modification
  • G is an guanosine comprising a 2'-deoxy-2'-fluoro modification
  • G is an guanosine comprising a 2'-0-methyl modification
  • U is an uridine comprising a 2'-deoxy-2'-fiuoro modification
  • U is an uridine comprising a 2'-0-methyl modification
  • C is an cytidine comprising a 2'-deoxy-2'-fiuoro modification
  • C is an cytidine comprising a 2'-0-methyl modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-deoxy-2'-fiuoro modification
  • Y for each occurrence, independently, is a nucleotide comprising a 2'-0-methyl modification.
  • L has the structure of LI :
  • R is R 3 and n is 2.
  • L has the structure of LI .
  • L has the structure of LI .
  • L has the structure of LI .
  • L has the structure of LI .
  • L has the structure of LI .
  • L has the structure of LI .
  • L has the structure of L2:
  • R is R 3 and n is 2.
  • L has the structure of L2. In an embodiment of the structure of formula (III), L has the structure of L2. In an embodiment of the structure of formula (IV), L has the structure of L2. In an embodiment of the structure of formula (V), L has the structure of L2. In an embodiment of the structure of formula (VI), L has the structure of L2. In an embodiment of the structure of formula (VII), L has the structure of L2. Delivery System
  • a delivery system for therapeutic nucleic acids having the structure of formula (VIII):
  • L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, wherein formula (VIII) optionally further comprises one or more branch point B, and one or more spacer S; wherein B is independently for each occurrence a polyvalent organic species or derivative thereof; S is independently for each occurrence selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; each cNA, independently, is a carrier nucleic acid comprising one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8.
  • L is an ethylene glycol chain. In another embodiment of the delivery system, L is an alkyl chain. In another embodiment of the delivery system, L is a peptide. In another embodiment of the delivery system, L is RNA. In another embodiment of the delivery system, L is DNA. In another embodiment of the delivery system, L is a phosphate. In another embodiment of the delivery system, L is a phosphonate. In another embodiment of the delivery system, L is a phosphoramidate. In another embodiment of the delivery system, L is an ester. In another embodiment of the delivery system, L is an amide. In another embodiment of the delivery system, L is a triazole.
  • S is an ethylene glycol chain. In another embodiment, S is an alkyl chain. In another embodiment of the delivery system, S is a peptide. In another embodiment, S is RNA. In another embodiment of the delivery system, S is DNA. In another embodiment of the delivery system, S is a phosphate. In another embodiment of the delivery system, S is a phosphonate. In another embodiment of the delivery system, S is a phosphoramidate. In another embodiment of the delivery system, S is an ester. In another embodiment, S is an amide. In another embodiment, S is a triazole.
  • n is 2. In another embodiment of the delivery system, n is 3. In another embodiment of the delivery system, n is 4. In another embodiment of the delivery system, n is 5. In another embodiment of the delivery system, n is 6. In another embodiment of the delivery system, n is 7. In another embodiment of the delivery system, n is 8.
  • each cNA comprises >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% chemically-modified nucleotides.
  • the compound of formula (VIII) has a structure selected from formulas (VIII-l )-(VIII-9) of Table 3 :
  • the compound of formula (VIII) is the structure of formula (VIII-
  • the compound of formula (VIII) is the structure of formula (VIII-2). In an embodiment, the compound of formula (VIII) is the structure of formula (VIII-3). In an embodiment, the compound of formula (VIII) is the structure of formula (VIII-4). In an embodiment, the compound of formula (VIII) is the structure of formula (VIII-5). In an embodiment, the compound of formula (VIII) is the structure of formula (VIII-6). In an embodiment, the compound of formula (VIII) is the structure of formula (VIII-7). In an embodiment, the compound of formula (VIII) is the structure of formula (VIII-8). In an embodiment, the compound of formula (VIII) is the structure of formula (VIII-9).
  • each cNA independently comprises at least 15 contiguous nucleotides. In an embodiment, each cNA independently consists of chemically-modified nucleotides.
  • the delivery system further comprises n therapeutic nucleic acids (NA), wherein each NA is hybridized to at least one cNA.
  • NA therapeutic nucleic acids
  • the delivery system is comprised of 2 NAs.
  • the delivery system is comprised of 3 NAs.
  • the delivery system is comprised of 4 NAs.
  • the delivery system is comprised of 5 NAs.
  • the delivery system is comprised of 6 NAs.
  • the delivery system is comprised of 7 NAs.
  • the delivery system is comprised of 8 NAs.
  • each NA independently comprises at least 16 contiguous nucleotides. In an embodiment, each NA independently comprises 16-20 contiguous nucleotides. In an embodiment, each NA independently comprises 16 contiguous nucleotides. In another embodiment, each NA independently comprises 17 contiguous nucleotides. In another embodiment, each NA independently comprises 18 contiguous nucleotides. In another embodiment, each NA independently comprises 19 contiguous nucleotides. In another embodiment, each NA independently comprises 20 contiguous nucleotides.
  • each NA comprises an unpaired overhang of at least 2 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 3 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 4 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 5 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 6 nucleotides. In an embodiment, the nucleotides of the overhang are connected via phosphorothioate linkages.
  • each NA is selected from the group consisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, or guide RNAs.
  • each NA is a DNA.
  • each NA is a siRNA.
  • each NA is an antagomiR.
  • each NA is a miRNA.
  • each NA is a gapmer.
  • each NA is a mixmer.
  • each NA independently, is a guide RNA.
  • each NA is the same. In an embodiment, each NA is not the same.
  • the delivery system further comprising n therapeutic nucleic acids
  • (NA) has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein.
  • the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein further comprising 2 therapeutic nucleic acids (NA).
  • the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein further comprising 3 therapeutic nucleic acids (NA).
  • the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein further comprising 4 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein further comprising 5 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein further comprising 6 therapeutic nucleic acids (NA).
  • the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein further comprising 7 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII), and embodiments thereof described herein further comprising 8 therapeutic nucleic acids (NA).
  • the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII) further comprising a linker of structure LI or L2 wherein R is R 3 and n is 2.
  • the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII) further comprising a linker of structure LI wherein R is R 3 and n is 2.
  • the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), (VII) further comprising a linker of structure L2 wherein R is R 3 and n is 2.
  • the target of delivery is selected from the group consisting of: brain, liver, skin, kidney, spleen, pancreas, colon, fat, lung, muscle, and thymus.
  • the target of delivery is the brain.
  • the target of delivery is the striatum of the brain.
  • the target of delivery is the cortex of the brain.
  • the target of delivery is the striatum of the brain.
  • the target of delivery is the liver.
  • the target of delivery is the skin.
  • the target of delivery is the kidney.
  • the target of delivery is the spleen.
  • the target of delivery is the pancreas.
  • the target of delivery is the colon. In one embodiment, the target of delivery is the fat. In one embodiment, the target of delivery is the lung. In one embodiment, the target of delivery is the muscle. In one embodiment, the target of delivery is the thymus. In one embodiment, the target of delivery is the spinal cord.
  • nucleic acids refers to RNA or DNA molecules consisting of a chain of ribonucleotides or deoxyribonucleotides, respectively.
  • therapeutic nucleic acid refers to a nucleic acid molecule (e.g. , ribonucleic acid) that has partial or complete complementarity to, and interacts with, a disease-associated target mRNA and mediates silencing of expression of the mRNA.
  • carrier nucleic acid refers to a nucleic acid molecule (e.g. , ribonucleic acid) that has sequence complementarity with, and hybridizes with, a therapeutic nucleic acid.
  • 3 'end refers to the end of the nucleic acid that contains an unmodified hydroxyl group at the 3 ' carbon of the ribose ring.
  • the term "5 'end” refers to the end of the nucleic acid that contains a phosphate group attached to the 5 ' carbon of the ribose ring.
  • nucleoside refers to a molecule made up of a heterocyclic base and its sugar.
  • nucleotide refers to a nucleoside having a phosphate group on its 3' or 5' sugar hydroxy! group.
  • siRNA refers to small interfering RNA duplexes that induce the RNA interference (RNAi) pathway. siRNA molecules can vary in length
  • RNA includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
  • antisense strand refers to the strand of the siRNA duplex that contains some degree of complementarity to the target gene.
  • sense strand refers to the strand of the siRNA duplex that contains complementarity to the antisense strand.
  • nucleotide analog or altered nucleotide or altered nucleotide refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides.
  • exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function.
  • positions of the nucleotide which may be derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc. ; the 6 position, e.g. , 6-(2-amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g. , 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc.
  • Nucleotide analogs also include deaza nucleotides, e.g.
  • O- and N-modified nucleotides e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art
  • other heterocyclically modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.
  • Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides.
  • the 2' OH-group may be replaced by a group selected from H, OR, R, F, CI, Br, I, SH, SR, NH2, NHR, NR 2 , COOR, or OR, wherein R is substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc.
  • Other possible modifications include those described in U. S. Pat. Nos. 5,858,988, and 6,291,438.
  • metabolic stabilized refers to RNA molecules that contain ribonucleotides that have been chemically modified from 2'-hydroxyl groups to 2'-0- methyl groups.
  • phosphorothioate refers to the phosphate group of a nucleotide that is modified by substituting one or more of the oxygens of the phosphate group with sulfur.
  • ethylene glycol chain refers to a carbon chain with the formula ((CH 2 OH) 2 ).
  • alkyl chain refers to an acyclic unsaturated hydrocarbon chain.
  • an “alkyl chain” includes but is not limited to straight chain, branch chain, and cyclic unsaturated hydrocarbon groups.
  • amide refers to an alkyl or aromatic group that is attached to an amino-carbonyl functional group.
  • nucleoside and “internucleotide” refer to the bonds between nucleosides and nucleotides, respectively.
  • triazol refers to heterocyclic compounds with the formula (C 2 H 3 N 3 ), having a five-membered ring of two carbons and three nitrogens, the positions of which can change resulting in multiple isomers.
  • terminal group refers to the group at which a carbon chain or nucleic acid ends.
  • lipophilic amino acid refers to an amino acid comprising a hydrophobic moiety (e.g., an alkyl chain or an aromatic ring).
  • antiagomiRs refers to nucleic acids that can function as inhibitors of miRNA activity.
  • glycomers refers to chimeric antisense nucleic acids that contain a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage.
  • the deoxynucleotide block is flanked by ribonucleotide monomers or
  • ribonucleotide monomers containing modifications refers to nucleic acids that are comprised of a mix of locked nucleic acids (LNAs) and DNA.
  • LNAs locked nucleic acids
  • guide RNAs refers to refers to nucleic acids that have sequence complementarity to a specific sequence in the genome immediately or 1 base pair upstream of the protospacer adjacent motif (PAM) sequence as used in CRISPR/Cas9 gene editing systems.
  • PAM protospacer adjacent motif
  • target of delivery refers to the organ or part of the body that is desired to deliver the branched oligonucleotide compositions to.
  • Di-siRNA refers to a molecule of the present invention that comprises a branched oligonucleotide structure and contains siRNA molecules as the therapeutic nucleic acids.
  • amino acid refers to a molecule containing amine and carboxyl functional groups and a side chain (R) specific to the amino acid.
  • amino acid has a structure of the formula:
  • amino acid may refer to a component residue of a peptide or protein having a structure of the formula:
  • the amino acid is chosen from the group of proteinogenic amino acids.
  • the amino acid is an L-amino acid or a D-amino acid.
  • the amino acid is a synthetic amino acid (e.g., a beta-amino acid).
  • internucleotide linkages provided herein comprising, e.g., phosphodiester and phosphorothioate, comprise a formal charge of -1 at physiological pH, and that said formal charge will be balanced by a cationic moiety, e.g., an alkali metal such as sodium or potassium, an alkali earth metal such as calcium or magnesium, or an ammonium or guanidinium ion.
  • a cationic moiety e.g., an alkali metal such as sodium or potassium, an alkali earth metal such as calcium or magnesium, or an ammonium or guanidinium ion.
  • the phosphate group of the nucleotide may also be modified, e.g. , by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2): 117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 1 1(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr.
  • Certain of the above-referenced modifications e.g., phosphate group modifications preferably decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.
  • a method for selectively delivering a nucleic acid as described herein to a particular organ in a patient comprising administering to the patient a branched oligonucleotide as described herein, such that the nucleic acid is delivered selectively.
  • the organ is the liver.
  • the organ is the kidneys.
  • the organ is the spleen.
  • the organ is the heart.
  • the organ is the brain.
  • compositions described herein promote simple, efficient, non-toxic delivery of metabolically stable oligonucleotides (e.g., siRNA), and promote potent silencing of therapeutic targets in a range of tissues in vivo.
  • metabolically stable oligonucleotides e.g., siRNA
  • Di-siRNA distributes throughout the injected hemisphere of the mouse brain following intrastriatal injection. While a single non conjugated siRNA can silence mRNA in primary neurons, the Di-siRNA structure is essential for enhanced tissue distribution and tissue retention of modified oligo nucleotides. Other conjugates such as cholesterol, although retained, show a steep gradient of diffusion away from the site of injection. The subtle hydrophobicity of the two single stranded phosphorothioated tails supports tissue retention while also allowing for widespread and uniform distribution throughout the ipsilateral hemisphere of the injected brain.
  • Di-siRNA shows a very unique cellular distribution when injected intrastriatally into the brain. Fluorescently labeled Di-siRNA appears to localize preferentially with neurons in the cortex. This selective feature is specific to these compounds and is not true for other siRNA conjugates such as cholesterol which show no cell type preference.
  • Di-siRNA shows localization to fiber tracts in the striatum but is present within neuronal cell bodies in the cortex. Movement to the cortex may be through diffusion or may be the result of retrograde transport via striatal fiber tracts. The theory that retrograde transport is partially responsible is supported by the fact that some areas of the cortex show full neuronal penetration while neurons in adjacent areas show no Di-siRNA association.
  • Di-siRNA A single therapeutically relevant brain injection of Di-siRNA results in widespread distribution of Di-siRNA throughout the brain.
  • the level of distribution demonstrated in Figures 31 -32 is unprecedented in the prior art and shows that Di-siRNAs are a promising therapeutic delivery system.
  • Di-siRNA shows widespread distribution throughout the body following a single intravenous injection. As shown in Figure 37, significant levels of Di-siRNAs were detected in mouse liver, skin, brain, kidney, spleen, pancreas, colon, fat, lung, muscle, and thymus. The finding that Di-siRNAs are present in the brain following intravenous injection also demonstrates that the Di-siRNA structures efficiently cross the blood-brain barrier.
  • compounds of the invention promote about 90% striatal silencing and about 65% cortical silencing in vivo in brain with a single injection, with no indication of toxicity. In some embodiments, compounds of the invention exhibit about 60% silencing throughout all regions of the spinal cord with intrathecal injection.
  • Di-siRNA Single injection of Di-siRNA induces robust silencing in both the striatum and cortex of mouse brain. This level of efficacy has never been demonstrated previously for non- conjugated siRNAs. Although Di-siRNA appears visually associated with fiber tracts in striatum, the efficacy observed clearly indicates that striatal neurons are internalizing Di- siRNA to a significant degree. In experiments, intrastriatal injection 2 nmols Di-siRNA (4 nmols of corresponding antisense HTT strand). Animals sacrificed 7 days post-injection. Tissue punches taken from the 300 um brain slices from the striatum and cortex.
  • Di-siRNA antisense strands present in different brain regions, liver, and kidney were quantified using Cy3 -labeled complimentary PNA to hybridize to the strand and HPLC to quantify ng of oligo per mg of tissue. aCSF - Artificial CSF.
  • Di-siRNA shows equal efficacy relative to a single siRNA duplex following lipid-mediated transfection in HeLa cells, indicating that RISC loading is not hindered by the tethering of two siRNA duplexes to a linker.
  • Di-siRNA is not efficacious in HeLa cells without transfection, however in primary cortical neurons, one phosphorothiated tail is enough to induce at least 60% silencing, suggesting that phosphorothiation is an effective method for delivering siRNA to primary neurons, without formulation.
  • Di-siRNA induces robust silencing in both the striatum and cortex of mouse brain.
  • a 63X image of pyrimidal neurons containing Cy3-labeled Di-branched oligo shows that Di-siRNA is visually associated with fiber tracts in the striatum, the efficacy observed clearly indicates that striatal neurons are internalizing Di-siRNA to a significant degree.
  • Di-siRNA shows robust and even silencing throughout the spinal cord following intrathecal injection.
  • a single injection of Di-siRNA in the lumbar region of the spinal cord silences mRNA to the same degree in the cervical, thoracic and lumbar regions indicating even and long range distribution.
  • Di-siRNA labeled with Cy3 induces robust silencing in both the striatum and cortex of mouse brain.
  • the mRNA expression levels show that the addition of Cy3 to the branched oligonucleotide compositions enhances silencing as compared to the unlabeled Di-siRNA.
  • a single intrastriatal injection resulted in silencing in both the cortex and striatum of mouse brain but did not result in any significant silencing in the liver or kidney. This demonstrates that branched oligonucleotides can specifically target an organ of interest.
  • a single injection of Di-siRNA continues to maintain robust silencing two weeks after the inj ection in both the striatum and cortex of mouse brain. Di- siRNA is stable and effective for at least two weeks in vivo.
  • Di- siRNA induces significant silencing in multiple areas of the brain. This is the first example of widespread siRNA silencing in the brain following a single therapeutically relevant injection.
  • an RNA silencing agent (or any portion thereof) of the invention as described supra may be modified such that the activity of the agent is further improved.
  • the RNA silencing agents described in above may be modified with any of the modifications described infra.
  • the modifications can, in part, serve to further enhance target discrimination, to enhance stability of the agent (e.g., to prevent degradation), to promote cellular uptake, to enhance the target efficiency, to improve efficacy in binding (e.g. , to the targets), to improve patient tolerance to the agent, and/or to reduce toxicity.
  • the RNA silencing agents of the invention may be substituted with a destabilizing nucleotide to enhance single nucleotide target discrimination (see U. S. application Ser. No. 11/698,689, filed Jan. 25, 2007 and U. S. Provisional Application No. 60/762,225 filed Jan. 25, 2006, both of which are incorporated herein by reference).
  • a modification may be sufficient to abolish the specificity of the RNA silencing agent for a non-target mRNA (e.g. wild-type mRNA), without appreciably affecting the specificity of the RNA silencing agent for a target mRNA (e.g. gain-of-function mutant mRNA).
  • the RNA silencing agents of the invention are modified by the introduction of at least one universal nucleotide in the antisense strand thereof.
  • Universal nucleotides comprise base portions that are capable of base pairing indiscriminately with any of the four conventional nucleotide bases (e.g. A, G, C, U).
  • a universal nucleotide is preferred because it has relatively minor effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA.
  • Exemplary universal nucleotide include those having an inosine base portion or an inosine analog base portion selected from the group consisting of deoxyinosine (e.g.
  • the universal nucleotide is an inosine residue or a naturally occurring analog thereof.
  • the RNA silencing agents of the invention are modified by the introduction of at least one destabilizing nucleotide within 5 nucleotides from a specificity-determining nucleotide (i. e. , the nucleotide which recognizes the disease-related polymorphism).
  • the destabilizing nucleotide may be introduced at a position that is within 5, 4, 3, 2, or 1 nucleotide(s) from a specificity-determining nucleotide.
  • the destabilizing nucleotide is introduced at a position which is 3 nucleotides from the specificity-determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destablilizing nucleotide and the specificity-determining nucleotide).
  • the destabilizing nucleotide may be introduced in the strand or strand portion that does not contain the specificity-determining nucleotide.
  • the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity- determining nucleotide.
  • the RNA silencing agents of the invention may be altered to facilitate enhanced efficacy and specificity in mediating RNAi according to asymmetry design rules (see U. S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705).
  • Such alterations facilitate entry of the antisense strand of the siRNA (e.g. , a siRNA designed using the methods of the invention or an siRNA produced from a shRNA) into RISC in favor of the sense strand, such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing.
  • RNA silencing agent Preferably the asymmetry of an RNA silencing agent is enhanced by lessening the base pair strength between the antisense strand 5' end (AS 5') and the sense strand 3' end (S 3') of the RNA silencing agent relative to the bond strength or base pair strength between the antisense strand 3' end (AS 3') and the sense strand 5' end (S '5) of said RNA silencing agent.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion than between the 3' end of the first or antisense strand and the 5' end of the sense strand portion.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion.
  • the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A: A, C:C and U:U.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a rare nucleotide, e.g., inosine (I).
  • the base pair is selected from the group consisting of an I:A, I:U and I:C.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a modified nucleotide.
  • the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6- diamino-A.
  • RNA silencing agents of the present invention can be modified to improve stability in serum or in growth medium for cell cultures.
  • the 3'-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNA interference.
  • the invention features RNA silencing agents that include first and second strands wherein the second strand and/or first strand is modified by the substitution of internal nucleotides with modified nucleotides, such that in vivo stability is enhanced as compared to a corresponding unmodified RNA silencing agent.
  • an "internal" nucleotide is one occurring at any position other than the 5' end or 3' end of nucleic acid molecule, polynucleotide or oligonucleotide.
  • An internal nucleotide can be within a single-stranded molecule or within a strand of a duplex or double-stranded molecule.
  • the sense strand and/or antisense strand is modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet another embodiment, the sense strand and/or antisense strand is modified by the substitution of all of the internal nucleotides.
  • the RNA silencing agents may contain at least one modified nucleotide analogue.
  • the nucleotide analogues may be located at positions where the target-specific silencing activity, e.g., the RNAi mediating activity or translational repression activity is not substantially effected, e.g. , in a region at the 5'-end and/or the 3'-end of the siRNA molecule.
  • the ends may be stabilized by incorporating modified nucleotide analogues.
  • Exemplary nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g. , of phosphothioate group.
  • the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or ON, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.
  • the modifications are 2'-fluoro, 2'-amino and/or 2'-thio modifications.
  • Particularly preferred modifications include 2'-fluoro-cytidine, 2'-fluoro- uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'- amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and/or 5-amino- allyl-uridine.
  • the 2'-fluoro ribonucleotides are every uridine and cytidine. Additional exemplary modifications include 5 -bromo -uridine, 5-iodo-uridine, 5- methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro- cytidine, and 5-fluoro-uridine. 2'-deoxy-nucleotides and 2'-Ome nucleotides can also be used within modified RNA-silencing agents moities of the instant invention.
  • Additional modified residues include, deoxy-abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin.
  • the 2' moiety is a methyl group such that the linking moiety is a 2'-0-methyl oligonucleotide.
  • the RNA silencing agent of the invention comprises Locked Nucleic Acids (LNAs).
  • LNAs comprise sugar-modified nucleotides that resist nuclease activities (are highly stable) and possess single nucleotide discrimination for mRNA (Elmen et al. , Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21 :74-81). These molecules have 2'- 0,4'-C-ethylene-bridged nucleic acids, with possible modifications such as 2'-deoxy-2"- fluorouridine.
  • LNAs increase the specificity of oligonucleotides by constraining the sugar moiety into the 3'-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10 °C per base.
  • the RNA silencing agent of the invention comprises Peptide Nucleic Acids (PNAs).
  • PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone which is highly resistant to nuclease digestion and imparts improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500).
  • nucleobase-modified ribonucleotides i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.
  • Bases may be modified to block the activity of adenosine deaminase.
  • modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g. , 5 -(2-amino )propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g.
  • cross-linking can be employed to alter the pharmacokinetics of the RNA silencing agent, for example, to increase half-life in the body.
  • the invention includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked.
  • the invention also includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3' terminus) to another moiety (e.g. a non- nucleic acid moiety such as a peptide), an organic compound (e.g. , a dye), or the like).
  • Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.
  • Other exemplary modifications include: (a) 2' modification, e.g. , provision of a 2' OMe moiety on a U in a sense or antisense strand, but especially on a sense strand, or provision of a 2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); (b) modification of the backbone, e.g., with the replacement of an 0 with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; e.g., with the replacement of a P with an S; (c) replacement of the U with a C5 amino linker; (d) replacement of an A with a G (s) 2'
  • Exemplary embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
  • Yet other exemplary modifications include the use of a methylated P in a 3' overhang, e.g., at the 3' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3' overhang, e.g., at the 3' terminus; modification with a 3' alkyl; modification with an abasic pyrrolidone in a 3' overhang, e.g., at the 3' terminus; modification with naproxen, ibuprofen, or other moieties which inhibit degradation at the 3' terminus
  • a compound of the invention may be modified with chemical moieties, for example, to enhance cellular uptake by target cells (e.g., neuronal cells).
  • target cells e.g., neuronal cells
  • the invention includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3' terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like.
  • the conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.
  • a compound of the invention is conjugated to a lipophilic moiety.
  • the lipophilic moiety is a ligand that includes a cationic group.
  • the lipophilic moiety is attached to one or both strands of an siRNA.
  • the lipophilic moiety is attached to one end of the sense strand of the siRNA.
  • the lipophilic moiety is attached to the 3' end of the sense strand.
  • the lipophilic moiety is selected from the group consisting of cholesterol, vitamin D, DHA, DHAg2, EPA, vitamin E, vitamin K, vitamin A, folic acid, or a cationic dye (e.g., Cy3).
  • RNA silencing agent tethered to a compound of the invention.
  • a ligand tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism.
  • Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting.
  • a tethered ligand can include one or more modified bases or sugars that can function as inter calators. These are preferably located in an internal region, such as in a bulge of RNA silencing agent/target duplex.
  • the intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound.
  • a polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings.
  • the universal bases described herein can be included on a ligand.
  • the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid.
  • the cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin- A2, or bleomycin- B2), pyrene, phenanthroline (e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr- lys tripeptide), or metal ion chelating group.
  • a bleomycin e.g., bleomycin-A5, bleomycin- A2, or bleomycin- B2
  • phenanthroline e.g., O-phenanthroline
  • polyamine e.g., a tripeptide (e.g., lys-tyr- lys tripeptide), or metal ion chelating group.
  • the metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III).
  • a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region.
  • 1 ,8-dimethyl-l, 3,6,8, 10, 13-hexaazacyclotetradecane can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage.
  • a tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity.
  • Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine.
  • Use of an acridine analog can increase sequence specificity.
  • neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity.
  • an acridine analog, neo-5-acridine has an increased affinity for the HIV Rev-response element (RRE).
  • the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent.
  • the amine group on the amino acid is exchanged for a guanidine group.
  • Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent.
  • a tethered ligand can be a poly- arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.
  • Exemplary ligands are coupled, preferably covalently, either directly or indirectly via an intervening tether, to a ligand-conjugated carrier.
  • the ligand is attached to the carrier via an intervening tether.
  • a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
  • Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases.
  • Lipophiles examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.
  • steroids e.g., uvaol, hecigenin, diosgenin
  • terpenes e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid
  • vitamins e.g., folic acid, vitamin A, biotin,
  • Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid.
  • HSA human serum albumin
  • LDL low-density lipoprotein
  • globulin carbohydrate
  • carbohydrate e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid
  • amino acid or a lipid.
  • the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.
  • polyamino acids examples include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L- glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine.
  • PLL polylysine
  • poly L-aspartic acid poly L- glutamic acid
  • styrene-maleic acid anhydride copolymer poly(L-lactide-co-glycolied) copolymer
  • divinyl ether-maleic anhydride copolymer divinyl ether-
  • polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
  • Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a cell or tissue targeting agent e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N- acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
  • ligands include dyes, intercalating agents (e.g. acridines and substituted acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides, guanidium aminoglycodies, artificial endonucleases (e.g.
  • intercalating agents e.g. acridines and substituted acridines
  • cross-linkers e.g. psoralene, mitomycin C
  • porphyrins TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine, phen
  • EDTA lipophilic molecules, e.g, cholesterol (and thio analogs thereof), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., C 10 , Cn, C 12 , C 13 , C 14 , C15, Ci6, Cn, Ci8, Ci9, or C 20 fatty acids) and ethers thereof, e.g., C 10 , Cn, C 12 , C 13 , C 14 , C 15 , C 16 , Cn, Ci 8 , Ci , or C2 0 alkyl; e.g., l,3-bis-0(hexadecyl)glycerol, 1, 3 -bis-O(octaadecyl) glycerol),
  • biotin e.g., aspirin, naproxen, vitamin E, folic acid
  • transport/absorption facilitators e.g., aspirin, naproxen, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.
  • Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
  • Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
  • the ligand can be a substance, e.g., a drug, which can increase the uptake of the RNA silencing agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments.
  • the drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
  • the ligand can increase the uptake of the RNA silencing agent into the cell by activating an inflammatory response, for example.
  • ligands that would have such an effect include tumor necrosis factor alpha (TNFa), interleukin-1 beta, or gamma interferon.
  • the ligand is a lipid or lipid-based molecule.
  • a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA).
  • HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body.
  • the target tissue can be the liver, including parenchymal cells of the liver.
  • Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used.
  • a lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
  • a lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
  • the lipid based ligand binds HSA.
  • a lipid-based ligand can bind HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
  • the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney.
  • Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
  • the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g. , a proliferating cell.
  • a target cell e.g. , a proliferating cell.
  • vitamins include vitamin A, E, and K.
  • Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells.
  • the ligand is a cell-permeation agent, preferably a helical cell- permeation agent.
  • the agent is amphipathic.
  • An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids.
  • the helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
  • the ligand can be a peptide or peptidomimetic.
  • 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 attachment of peptide and peptidomimetics to oligonucleotide agents can affect pharmacokinetic distribution of the RNA silencing agent, such as by enhancing cellular recognition and absorption.
  • the peptide or peptidomimetic moiety 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.
  • a peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe).
  • the peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide.
  • the peptide moiety can be an L-peptide or D- peptide.
  • the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).
  • a peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one- compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991).
  • the peptide or peptidomimetic tethered to an RNA silencing agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine- aspartic acid (RGD)-peptide, or RGD mimic.
  • a peptide moiety can range in length from about 5 amino acids to about 40 amino acids.
  • the peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized EXAMPLES
  • Di-siRNAs used in the in vitro and in vivo efficacy evaluation were synthesized as follows. As shown in FIG. 2, triethylene glycol was reacted with acrylonitrile to introduce protected amine functionality. A branch point was then added as a tosylated solketal, followed by reduction of the nitrile to yield a primary amine which was then attached to vitamin D (calciferol) through a carbamate linker. The ketal was then hydrolyzed to release the cis-diol which was selectively protected at the primary hydroxyl with dimethoxytrityl (DMTr) protecting group, followed by succinylation with succinic anhydride.
  • DMTr dimethoxytrityl
  • the mono-phosphoamidate linker approach involves the following steps: Mono-azide tetraethylene glycol has a branch point added as a tosylated solketal. The ketal is then removed to release the cis-diol which is selectively protected at the primary hydroxyl with dimethoxytrityl (DMTr) protecting group, followed by reduction of the azide by triphenylphosphine to a primary amine, which is immediately protected with a monomethoxy trityl (MMTr) protecting group. The remaining hydroxyl is succinylated with succinic anhydride and coulped to solid support (LCAA CPG).
  • DMTr dimethoxytrityl
  • MMTr monomethoxy trityl
  • Oligonucleotide synthesis and deprotection affords one main product the, the di-siRNA with a phosphate and phosphoamidate linkage.
  • This example highlights an alternative and direct route of synthesis to produce solely the phosphate and phosphoamidate linker.
  • the di-phosphoate linker approach involves the following steps: Starting from a solketal-modified teraethylene glycol, the ketal is removed and the two primary hydroxyls are selectively protected with dimethoxy trityl (DMTr). The remaining hydroxyl is extended in length with a silyl protected 1-bromoethanol. The TBDMS is removed, succinylated and attached to solid support. This is followed by solid phase oligonucleotide synthesis and deprotection, producing the Di-siRNA with the diphosphate containing linker.
  • DMTr dimethoxy trityl
  • the Di-siRNA product showed dramatically increased uptake in the injected hemisphere of the mouse brain compared to the other three by-products (Fig 22). Of the four by-products resulting from the chemical synthesis reaction, the Di-siRNA shows both efficient gene-silencing and high levels of cellular uptake in vivo.
  • Di-siRNAs targeting Htt were transfected into HeLa cells using a lipid-mediated delivery system. HeLa cells were transfected with branched oligonucleotides at varying concentrations using RNAiMax. HTT mRNA expression was measured 72 hours after transfection. The Di- siRNAs caused significant silencing of the HTT gene, similar to the effect resulting from single siRNA duplex in HeLa cells (FIG. 10).
  • Htt-Di-siRNAs were treated passively with Htt-Di-siRNAs at varying concentrations for one week. HTT mRNA expression was measured and normalized to the housekeeping gene PPIB. As shown in FIG. 10, the Di-siRNA structure led to significant silencing of the Htt gene, showing that the Di-branched siRNA structure is efficiently delivered to neurons without the lipid formulation. This demonstrates that the Di- branched structure of the siRNA complex does not hinder RISC loading and the gene silencing effects of known effective siRNAs.
  • Di-HTT-Cy3 was delivered to mice via intrastriatal (IS) injection.
  • Di-HTT-Cy3 localized to and accumulated throughout the injected hemisphere of the brain, whereas the single branch HTT-siRNA (tryethylene glycol conjugated siRNA (TEG-siRNA)) showed significantly lower accumulation in the injected hemisphere of the brain (FIG. 11).
  • TEG-siRNA tryethylene glycol conjugated siRNA
  • the double-branch structure of the Di-siRNAs significantly improves distribution and neuronal uptake when compared with the TEG-siRNA only; therefore it is likely that the size and/or the structure of the siRNA complex are important for efficacy.
  • the IS injection of Htt-Di-siRNAs leads to significant and stable depletion of Htt, which stays localized to the brain, this level of efficacy has never been demonstrated for non- conjugated siRNAs.
  • Di-HTT-Cy3 was delivered to mice via intrathecal (IT) injection in the lumbar region of the spinal cord. As shown in FIGS. 14 and 28-29, Di-HTT-Cy3 accumulated in the spinal cord one week post injection. IT injection also led to significant Htt mRNA silencing in the cervical, thoracic, and lumbar regions of the spinal cord one week post injection (FIG. 14). The IT injection of Di-HTT-Cy3 successfully led to significant gene silencing in the spinal cord.
  • Di-HTT-Cy3 was delivered to mice via intracerebroventricular (ICV) injection.
  • ICV intracerebroventricular
  • the Di-siRNAs accumulated throughout the brain at both two days and two weeks post injection (FIGS. 30-31).
  • ICV injection of Di-HTT-Cy3 also significantly silenced Htt mRNA and protein expression two weeks post injection (FIG. 32). Further experiments showed the ICV delivery did not result in significant toxicity two weeks post injection (Fig 33).
  • ICV injection of Di-HTT-Cy3 did result in significant gliosis in multiple areas of the brain, which is an expected result upon silencing of the Htt gene (FIG. 34).
  • the ICV injection directly administers the Di-siRNAs to the cerebrospinal fluid (CSF) in order to bypass the blood brain barrier and this injection is used to treat diseases of the brain.
  • CSF cerebrospinal fluid
  • This result is important for the therapeutic potential of branched oligonucleotides, as ICV injection is a therapeutically relevant injection for neurological diseases.
  • the efficacy and stability of the branched oligonucleotides following ICV administration demonstrates that the invention described herein could be utilized as therapy in a variety of hard to treat neurological diseases, including Huntington's disease.
  • Di- HTT-Cy3 was administered to mice via intravenous (IV) injection.
  • the mice were injected with 20 mg/kg Di-HTT-Cy3 and two consecutive days (total of 40 mg/kg) and were sacrificed 24 hours after the final injection.
  • the Di-siRNAs accumulated in multiple organs (including liver, kidney, spleen, pancreas, lung, fat, muscle, thymus, colon, and skin) following IV delivery.
  • the Di-siRNAs also accumulated in the brain, demonstrating the ability of the Di-siRNAs to cross the blood-brain barrier, an unprecedented result using therapeutic siRNAs.
  • the IV injection demonstrates that the Di- siRNA structure is effective and functional in a wide variety of cell types throughout the body.
  • DARPP32 protein was quantified by immunoblot. Artificial cerebrospinal fluid (aCSF) was used as a negative control. Neither IS nor ICV injection of high dose Di-HTT-Cy3 resulted in significant toxicity (FIGS. 27 and 33).
  • aCSF Artificial cerebrospinal fluid
  • GFAP protein levels were assessed following high dose of Di-HTT-Cy3.
  • Mice were treated with 2 nmols Di-HTT-Cy3 (4 nmols of corresponding antisense HTT strand) via IS or ICV injection. The animals were sacrificed 14 days after injection and tissue punches were taken from 300 ⁇ brain slices from different areas of the brain. GFAP protein was quantified by immunoblot. Artificial cerebrospinal fluid (aCSF) was used as a negative control. Artificial cerebrospinal fluid (aCSF) was used as a negative control. Both IS and ICV injection of high dose Di-HTT-Cy3 resulted in significant gliosis (FIGS. 27 and 34), however induction of gliosis is an expected result upon near complete silencing of the Huntingtin gene.
  • mice were treated with Di-HTT-Cy3 via IS, ICV, intrathecal, or IV injections as described above in Examples 7-10.
  • 2 nmols Di-HTT-Cy3 (4 nmols of corresponding antisense HTT strand) was injected and accumulation was quantified by using Cy3 -labeled peptide nucleic acids (PNAs) to hybridize to the sense strand.
  • PNAs Cy3 -labeled peptide nucleic acids
  • HPLC analysis was then used to quantify ng of Di-HTT-Cy3 per mg of tissue.
  • Artificial cerebrospinal fluid (aCSF) was used as a negative control.
  • mice were treated with Di-HTT-Cy3 via IS, ICV, intrathecal, or IV injections as described above in Examples 7-10.
  • 2 nmols Di-HTT-Cy3 (4 nmols of corresponding antisense HTT strand) was injected and silencing of Htt mRNA was quantified using Affymetrix Quantigene 2.0 as described in Coles, A. et al., A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo. Nucl Acid Ther. 26 (2), 86-92, 2015. Data was normalized to the housekeeping control, HPRT and artificial cerebrospinal fluid (aCSF) was used as a negative control.
  • aCSF artificial cerebrospinal fluid
  • a short hydrophobic alkylene or alkane (Hy) with an unprotected hydroxyl group (or amine) that can be phosphitylated with 2-Cyanoethoxy-bis(N,N- diisopropylamino)phosphine (or any other suitable phosphitylating reagent) is used to produce the corresponding lipophilic phosphoramidite.
  • These lipophilic phosphoramidites can be added to the terminal position of the branched oligonucleotide using conventional oligonucleotide synthesis conditions. This strategy is depicted in FIG. 44.
  • a short/small aromatic planar molecule that has an unprotected hydroxyl group with or without a positive charge (or amine) that can be phosphitylated with 2-Cyanoethoxy-bis(N,N-diisopropylamino)phosphine (or any other suitable phosphitylating reagent) is used to produce the corresponding aromatic hydrophobic phosphoramidite.
  • the aromatic moiety can have a positive charge.
  • short lipophilic peptides are made by sequential peptide synthesis either on solid support or in solution (the latter being described here).
  • the short (1-10) amino acid chain can contain positively charged or polar amino acid moieties as well, as any positive charge will reduce the overall net charge of the oligonucleotide, therefore increasing the hydrophobicity.
  • the peptide of appropriate length is made it should be capped with acetic anhydride or another short aliphatic acid to increase hydrophobicity and mask the free amine.
  • the carbonyl protecting group is then removed to allow for 3-aminopropan-l -ol to be coupled allowing a free hydroxyl (or amine) to be phosphitylated.
  • This amino acid phosphoramidite can then be added to the terminal 5 ⁇ position of the branched oligonucleotide using conventional oligonucleotide synthesis conditions. This strategy is depicted in FIG. 46.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Psychology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/US2017/015633 2016-01-31 2017-01-30 Branched oligonucleotides Ceased WO2017132669A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
EP17745083.0A EP3408391A4 (en) 2016-01-31 2017-01-30 BRANCHED OLIGONUCLEOTIDES
CA3011894A CA3011894A1 (en) 2016-01-31 2017-01-30 Branched oligonucleotides
AU2017210726A AU2017210726B2 (en) 2016-01-31 2017-01-30 Branched oligonucleotides
HK19101058.6A HK1258758A1 (zh) 2016-01-31 2017-01-30 分支的寡核苷酸
JP2018539903A JP7749201B6 (ja) 2016-01-31 2017-01-30 分岐オリゴヌクレオチド
JP2022200533A JP2023040012A (ja) 2016-01-31 2022-12-15 分岐オリゴヌクレオチド
AU2023206109A AU2023206109A1 (en) 2016-01-31 2023-07-18 Branched oligonucleotides
JP2024232308A JP2025066730A (ja) 2016-01-31 2024-12-27 分岐オリゴヌクレオチド

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201662289268P 2016-01-31 2016-01-31
US62/289,268 2016-01-31
US201662317113P 2016-04-01 2016-04-01
US62/317,113 2016-04-01

Publications (1)

Publication Number Publication Date
WO2017132669A1 true WO2017132669A1 (en) 2017-08-03

Family

ID=59399003

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2017/015633 Ceased WO2017132669A1 (en) 2016-01-31 2017-01-30 Branched oligonucleotides

Country Status (7)

Country Link
US (4) US10478503B2 (enExample)
EP (1) EP3408391A4 (enExample)
JP (3) JP7749201B6 (enExample)
AU (2) AU2017210726B2 (enExample)
CA (1) CA3011894A1 (enExample)
HK (1) HK1258758A1 (enExample)
WO (1) WO2017132669A1 (enExample)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019183000A1 (en) * 2018-03-19 2019-09-26 University Of Massachusetts Modified guide rnas for crispr genome editing
US20210115442A1 (en) * 2019-08-23 2021-04-22 University Of Massachusetts O-methyl rich fully stabilized oligonucleotides
US20210363523A1 (en) * 2020-03-18 2021-11-25 University Of Massachusetts Oligonucleotides for mapt modulation
CN113811311A (zh) * 2019-03-15 2021-12-17 马萨诸塞大学 用于组织特异性apoe调节的寡核苷酸
JP2022547790A (ja) * 2019-08-09 2022-11-16 ユニバーシティー オブ マサチューセッツ Ｓｎｐを標的とする化学修飾オリゴヌクレオチド
US11702659B2 (en) 2021-06-23 2023-07-18 University Of Massachusetts Optimized anti-FLT1 oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders
EP3833763A4 (en) * 2018-08-10 2023-07-19 University of Massachusetts MODIFIED OLIGONUCLEOTIDES FOR TARGETING SNPS
EP3946374A4 (en) * 2019-03-29 2023-07-19 University of Massachusetts OLIGONUCLEOTIDE-BASED MODULATION OF C9ORF72
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides
US12049627B2 (en) 2017-06-23 2024-07-30 University Of Massachusetts Two-tailed self-delivering siRNA
US12077755B2 (en) 2015-08-14 2024-09-03 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US12077758B2 (en) 2020-05-28 2024-09-03 University Of Massachusetts Oligonucleotides for SARS-CoV-2 modulation
US12173286B2 (en) 2015-04-03 2024-12-24 University Of Massachusetts Fully stabilized asymmetric siRNA
US12180477B2 (en) 2019-01-18 2024-12-31 University Of Massachusetts Dynamic pharmacokinetic-modifying anchors
US12258566B2 (en) 2020-02-28 2025-03-25 University Of Massachusetts Oligonucleotides for PRNP modulation
US12297430B2 (en) 2018-08-23 2025-05-13 University Of Massachusetts O-methyl rich fully stabilized oligonucleotides
EP4560019A1 (en) 2023-11-22 2025-05-28 Sylentis S.A.U. Sirna and compositions for prophylactic and therapeutic treatment of ocular retinal conditions
EP4560020A1 (en) 2023-11-22 2025-05-28 Sylentis S.A.U. Sirna and compositions for prophylactic and therapeutic treatment of ocular retinal conditions
US12365894B2 (en) 2019-09-16 2025-07-22 University Of Massachusetts Branched lipid conjugates of siRNA for specific tissue delivery
US12378554B2 (en) 2019-05-06 2025-08-05 University Of Massachusetts Anti-C9ORF72 oligonucleotides and related methods

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK3277815T3 (da) 2015-04-03 2021-12-13 Univ Massachusetts Oligonukleotidforbindelser til behandling af præeklampsi og andre angiogene lidelser
DK3277814T3 (da) 2015-04-03 2020-08-24 Univ Massachusetts Oligonukleotidforbindelser til målretning mod huntingtin-mrna
WO2018031933A2 (en) 2016-08-12 2018-02-15 University Of Massachusetts Conjugated oligonucleotides
WO2019136459A1 (en) 2018-01-08 2019-07-11 Iovance Biotherapeutics, Inc. Processes for generating til products enriched for tumor antigen-specific t-cells
US11713446B2 (en) 2018-01-08 2023-08-01 Iovance Biotherapeutics, Inc. Processes for generating TIL products enriched for tumor antigen-specific T-cells
KR20200119242A (ko) 2018-01-08 2020-10-19 이오반스 바이오테라퓨틱스, 인크. 종양 항원-특이적 t-세포에 대해 풍부화된 til 생성물의 생성 방법
AU2019265904A1 (en) 2018-05-11 2020-11-12 Wave Life Sciences Ltd. Oligonucleotide compositions and methods of use thereof
WO2020198509A2 (en) * 2019-03-26 2020-10-01 University Of Massachusetts Modified oligonucleotides with increased stability
CA3162845A1 (en) * 2019-12-23 2021-07-01 Anastasia Khvorova Oligonucleotides for tissue specific gene expression modulation
EP4121538A4 (en) * 2020-03-18 2024-07-31 University Of Massachusetts OLIGONUCLEOTIDES FOR SNCA MODULATION
AU2021244600A1 (en) 2020-03-26 2022-11-03 University Of Massachusetts Synthesis of modified oligonucleotides with increased stability
EP4125930A4 (en) * 2020-03-27 2025-06-04 University Of Massachusetts Dual-acting siRNA-based modulation of C9ORF72
WO2021216556A2 (en) * 2020-04-20 2021-10-28 University Of Massachusetts Oligonucleotides for msh3 modulation
US20240318174A1 (en) * 2021-02-08 2024-09-26 Ractigen Therapeutics Multi-Valent Oligonucleotide Agent and Methods of Use Thereof
JP2024512612A (ja) * 2021-03-24 2024-03-19 アタランタ セラピューティクス，インコーポレーテッド 二本鎖ｓｉＲＮＡを使用したミクログリア遺伝子サイレンシング
WO2022212208A1 (en) * 2021-03-29 2022-10-06 University Of Massachusetts Oligonucleotides for syngr-3 modulation
US20230193281A1 (en) * 2021-11-08 2023-06-22 University Of Massachusetts Oligonucleotides for sod1 modulation
WO2023114959A2 (en) * 2021-12-17 2023-06-22 University Of Massachusetts Oligonucleotides for mlh1 modulation
WO2025101484A1 (en) 2023-11-06 2025-05-15 Iovance Biotherapeutics, Inc. Treatment of endometrial cancers with tumor infiltrating lymphocyte therapies

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6489464B1 (en) * 1994-06-01 2002-12-03 Hybridon, Inc. Branched oligonucleotides as pathogen-inhibitory agents
US20060009409A1 (en) * 2002-02-01 2006-01-12 Woolf Tod M Double-stranded oligonucleotides
US20070004664A1 (en) * 2002-09-05 2007-01-04 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)

Family Cites Families (190)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US586935A (en) * 1897-07-20 Albert j
US4522811A (en) 1982-07-08 1985-06-11 Syntex (U.S.A.) Inc. Serial injection of muramyldipeptides and liposomes enhances the anti-infective activity of muramyldipeptides
US5240848A (en) 1988-11-21 1993-08-31 Monsanto Company Dna sequences encoding human vascular permeability factor having 189 amino acids
US5328470A (en) 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US5332671A (en) 1989-05-12 1994-07-26 Genetech, Inc. Production of vascular endothelial cell growth factor and DNA encoding same
US5219739A (en) 1989-07-27 1993-06-15 Scios Nova Inc. DNA sequences encoding bVEGF120 and hVEGF121 and methods for the production of bovine and human vascular endothelial cell growth factors, bVEGF120 and hVEGF121
US5194596A (en) 1989-07-27 1993-03-16 California Biotechnology Inc. Production of vascular endothelial cell growth factor
EP0500799B1 (en) 1989-11-16 1998-01-14 Duke University Particle mediated transformation of animal skin tissue cells
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5672697A (en) 1991-02-08 1997-09-30 Gilead Sciences, Inc. Nucleoside 5'-methylene phosphonates
US5252479A (en) 1991-11-08 1993-10-12 Research Corporation Technologies, Inc. Safe vector for gene therapy
US5587308A (en) 1992-06-02 1996-12-24 The United States Of America As Represented By The Department Of Health & Human Services Modified adeno-associated virus vector capable of expression from a novel promoter
JPH0641183A (ja) 1992-07-23 1994-02-15 Mitsubishi Kasei Corp オリゴヌクレオチド単分子膜
US5985558A (en) 1997-04-14 1999-11-16 Isis Pharmaceuticals Inc. Antisense oligonucleotide compositions and methods for the inibition of c-Jun and c-Fos
US5858988A (en) 1993-02-24 1999-01-12 Wang; Jui H. Poly-substituted-phenyl-oligoribo nucleotides having enhanced stability and membrane permeability and methods of use
US6291438B1 (en) 1993-02-24 2001-09-18 Jui H. Wang Antiviral anticancer poly-substituted phenyl derivatized oligoribonucleotides and methods for their use
EP0691979A1 (en) 1993-03-31 1996-01-17 Sanofi Novel 5'-substituted nucleosides and oligomers produced therefrom
TW404844B (en) 1993-04-08 2000-09-11 Oxford Biosciences Ltd Needleless syringe
WO1996003500A1 (en) 1994-07-26 1996-02-08 Ltt Institute Co., Ltd. Sustance with antiviral activity
US6383814B1 (en) 1994-12-09 2002-05-07 Genzyme Corporation Cationic amphiphiles for intracellular delivery of therapeutic molecules
WO1996033761A1 (en) 1995-04-28 1996-10-31 Medtronic, Inc. Intraparenchymal infusion catheter system
US6025335A (en) 1995-09-21 2000-02-15 Lipitek International, Inc. L-Nucleoside Dimer Compounds and therapeutic uses
US5684143A (en) 1996-02-21 1997-11-04 Lynx Therapeutics, Inc. Oligo-2'-fluoronucleotide N3'->P5' phosphoramidates
US5735814A (en) 1996-04-30 1998-04-07 Medtronic, Inc. Techniques of treating neurodegenerative disorders by brain infusion
US5898031A (en) 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
WO2005121370A2 (en) 2004-06-03 2005-12-22 Isis Pharmaceuticals, Inc. Oligomeric compounds that facilitate risc loading
US5849902A (en) * 1996-09-26 1998-12-15 Oligos Etc. Inc. Three component chimeric antisense oligonucleotides
US6177403B1 (en) 1996-10-21 2001-01-23 The Trustees Of The University Of Pennsylvania Compositions, methods, and apparatus for delivery of a macromolecular assembly to an extravascular tissue of an animal
US6042820A (en) 1996-12-20 2000-03-28 Connaught Laboratories Limited Biodegradable copolymer containing α-hydroxy acid and α-amino acid units
US6472375B1 (en) 1998-04-16 2002-10-29 John Wayne Cancer Institute DNA vaccine and methods for its use
US6242589B1 (en) 1998-07-14 2001-06-05 Isis Pharmaceuticals, Inc. Phosphorothioate oligonucleotides having modified internucleoside linkages
EP1016726A1 (en) 1998-12-30 2000-07-05 Introgene B.V. Gene therapy to promote angiogenesis
WO2001045746A2 (en) 1999-12-24 2001-06-28 Genentech, Inc. Methods and compositions for prolonging elimination half-times of bioactive compounds
US8273866B2 (en) 2002-02-20 2012-09-25 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
US8202979B2 (en) 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
WO2002081628A2 (en) 2001-04-05 2002-10-17 Ribozyme Pharmaceuticals, Incorporated Modulation of gene expression associated with inflammation proliferation and neurite outgrowth, using nucleic acid based technologies
CZ302719B6 (cs) 2000-12-01 2011-09-21 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Izolovaná molekula dvouretezcové RNA, zpusob její výroby a její použití
WO2003029459A2 (en) 2001-09-28 2003-04-10 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Micro-rna molecules
WO2003053363A2 (en) 2001-12-19 2003-07-03 Millennium Pharmaceuticals, Inc. Human diacylglycerol acyltransferase 2 (dgat2) family members and uses therefor
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20100240730A1 (en) 2002-02-20 2010-09-23 Merck Sharp And Dohme Corp. RNA Interference Mediated Inhibition of Gene Expression Using Chemically Modified Short Interfering Nucleic Acid (siNA)
US20050096284A1 (en) 2002-02-20 2005-05-05 Sirna Therapeutics, Inc. RNA interference mediated treatment of polyglutamine (polyQ) repeat expansion diseases using short interfering nucleic acid (siNA)
WO2003092594A2 (en) 2002-04-30 2003-11-13 Duke University Adeno-associated viral vectors and methods for their production from hybrid adenovirus and for their use
OA12890A (en) 2002-07-19 2006-10-13 Beth Israel Hospital Methods of diagnosing and treating pre-eclampsia or eclampsia.
US20050106731A1 (en) 2002-08-05 2005-05-19 Davidson Beverly L. siRNA-mediated gene silencing with viral vectors
US20040241854A1 (en) 2002-08-05 2004-12-02 Davidson Beverly L. siRNA-mediated gene silencing
WO2004041167A2 (en) 2002-10-30 2004-05-21 Theodora Calogeropoulou Antiprotozoal ring-substituted phospholipids
WO2004044136A2 (en) 2002-11-05 2004-05-27 Isis Pharmaceuticals, Inc. Compositions comprising alternating 2’-modified nucleosides for use in gene modulation
WO2006006948A2 (en) 2002-11-14 2006-01-19 Dharmacon, Inc. METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY
EP1560931B1 (en) 2002-11-14 2011-07-27 Dharmacon, Inc. Functional and hyperfunctional sirna
US7250496B2 (en) 2002-11-14 2007-07-31 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory genes and uses thereof
US7790867B2 (en) 2002-12-05 2010-09-07 Rosetta Genomics Inc. Vaccinia virus-related nucleic acids and microRNA
US6913890B2 (en) 2002-12-18 2005-07-05 Palo Alto Research Center Incorporated Process for preparing albumin protein conjugated oligonucleotide probes
US20080188429A1 (en) 2002-12-27 2008-08-07 Iyer Radhakrishnan P Synthetic siRNA compounds and methods for the downregulation of gene expression
US20040198640A1 (en) 2003-04-02 2004-10-07 Dharmacon, Inc. Stabilized polynucleotides for use in RNA interference
AU2004233092C9 (en) 2003-04-17 2010-10-28 Alnylam Pharmaceuticals, Inc. Modified iRNA agents
US7851615B2 (en) 2003-04-17 2010-12-14 Alnylam Pharmaceuticals, Inc. Lipophilic conjugated iRNA agents
US7750144B2 (en) 2003-06-02 2010-07-06 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of RNA silencing
AU2004252442B2 (en) 2003-06-02 2010-04-08 University Of Massachusetts Methods and compositions for enhancing the efficacy and specificity of FNAi
PT1633767T (pt) 2003-06-02 2019-02-27 Univ Massachusetts Métodos e composições para controlar a eficácia do silenciamento de arn
DE10326302A1 (de) 2003-06-11 2004-12-30 Gnothis Holding Sa Bi-Fluorophor-markierte Sonden zum Nachweis von Nukleinsäuren
US8680063B2 (en) 2003-09-12 2014-03-25 University Of Massachusetts RNA interference for the treatment of gain-of-function disorders
BRPI0413930A (pt) 2003-09-18 2006-10-24 Isis Pharmaceuticals Inc composto oligomérico ou sal farmaceuticamente aceitável deste, composição farmacêutica ou veterinária, métodos para inibir a expressão de eif4e em uma célula, tecido ou órgão, para diminuir a proliferação de uma célula em que eif4e é expressado, e para prevenir ou tratar uma condição ou doença métodos para prevenir ou diminuir a angiogênese, e o crescimento de tumor em um paciente, oligonucleotìdeo de anti-sentido, composição farmacêutica ou veterinária, e, uso de um composto oligomérico ou sal farmaceuticamente aceitável deste
WO2005060697A2 (en) 2003-12-19 2005-07-07 Chiron Corporation Cell transfecting formulations of small interfering rna, related compositions and methods of making and use
US20050176045A1 (en) 2004-02-06 2005-08-11 Dharmacon, Inc. SNP discriminatory siRNA
WO2005077333A2 (en) 2004-02-10 2005-08-25 University Of Florida Research Foundation, Inc. Gel-based delivery of recombinant adeno-associated virus vectors
EP2636739B1 (en) 2004-03-12 2014-12-10 Alnylam Pharmaceuticals Inc. iRNA agents targeting VEGF
KR101147147B1 (ko) 2004-04-01 2012-05-25 머크 샤프 앤드 돔 코포레이션 Ｒｎａ 간섭의 오프 타겟 효과 감소를 위한 변형된폴리뉴클레오타이드
MXPA06012076A (es) 2004-04-20 2007-01-25 Nastech Pharm Co Metodos y composiciones para mejorar el suministro de arn bicatenario o un acido nucleico hibrido bicatenario para regular la expresion genetica en celulas de mamifero.
EP1752536A4 (en) 2004-05-11 2008-04-16 Alphagen Co Ltd POLYNUCLEOTIDE CAUSING RNA INTERFERENCE AND METHOD OF REGULATING GENE EXPRESSION WITH THE USE OF THE SAME
EP1789553B1 (en) 2004-06-30 2014-03-26 Alnylam Pharmaceuticals Inc. Oligonucleotides comprising a non-phosphate backbone linkage
EP1828390B1 (en) 2004-12-15 2012-06-13 The University Of North Carolina At Chapel Hill Chimeric vectors
US20070161591A1 (en) 2005-08-18 2007-07-12 University Of Massachusetts Methods and compositions for treating neurological disease
EP1931780B1 (en) 2005-08-29 2016-01-06 Regulus Therapeutics Inc. Antisense compounds having enhanced anti-microrna activity
JP5111385B2 (ja) 2005-10-28 2013-01-09 アルナイラム ファーマシューティカルズ， インコーポレイテッド ハンチンチン遺伝子の発現を抑制するための組成物および方法
CA2628300C (en) 2005-11-02 2018-04-17 Protiva Biotherapeutics, Inc. Modified sirna molecules and uses thereof
JP2009514877A (ja) 2005-11-04 2009-04-09 エムディーアールエヌエー，インコーポレイテッド ｓｉＲＮＡの送達媒体としてのペプチド−ダイザ基質ＲＮＡ抱合体
US20070259827A1 (en) 2006-01-25 2007-11-08 University Of Massachusetts Compositions and methods for enhancing discriminatory RNA interference
US9198981B2 (en) 2006-02-01 2015-12-01 The University Of Kentucky Modulation of angiogenesis
US8362229B2 (en) 2006-02-08 2013-01-29 Quark Pharmaceuticals, Inc. Tandem siRNAS
WO2007094218A1 (ja) 2006-02-16 2007-08-23 Gifu University 修飾オリゴヌクレオチド
US20100184209A1 (en) 2006-02-17 2010-07-22 Dharmacon, Inc. Compositions and methods for inhibiting gene silencing by rna interference
WO2007112414A2 (en) 2006-03-27 2007-10-04 Isis Pharmaceuticals, Inc. Conjugated double strand compositions for use in gene modulation
CA2662704A1 (en) 2006-07-07 2008-01-10 University Of Massachusetts Rna silencing compositions and methods for the treatment of huntington's disease
US20080039415A1 (en) 2006-08-11 2008-02-14 Gregory Robert Stewart Retrograde transport of sirna and therapeutic uses to treat neurologic disorders
AU2007299705B2 (en) 2006-09-22 2012-09-06 Dharmacon, Inc. Duplex oligonucleotide complexes and methods for gene silencing by RNA interference
US8906874B2 (en) 2006-11-09 2014-12-09 Gradalis, Inc. Bi-functional shRNA targeting Stathmin 1 and uses thereof
CA2679342A1 (en) 2007-03-02 2008-09-12 Mdrna, Inc. Nucleic acid compounds for inhibiting hif1a gene expression and uses thereof
WO2008154482A2 (en) 2007-06-08 2008-12-18 Sirnaomics, Inc. Sirna compositions and methods of use in treatment of ocular diseases
US20110046206A1 (en) 2007-06-22 2011-02-24 Isis Pharmaceuticals, Inc. Double strand compositions comprising differentially modified strands for use in gene modulation
ES2617877T3 (es) 2007-08-27 2017-06-20 1Globe Health Institute Llc Composiciones de ARN interferente asimétrico y uso de las mismas
CN101199858A (zh) 2007-10-18 2008-06-18 广州拓谱基因技术有限公司 治疗眼科疾病的多靶点小干扰rna鸡尾酒制剂及制备方法
TW200927177A (en) 2007-10-24 2009-07-01 Nat Inst Of Advanced Ind Scien Lipid-modified double-stranded RNA having potent RNA interference effect
EP4074344A1 (en) 2007-12-04 2022-10-19 Arbutus Biopharma Corporation Targeting lipids
US9029524B2 (en) 2007-12-10 2015-05-12 California Institute Of Technology Signal activated RNA interference
US20110081362A1 (en) 2008-01-31 2011-04-07 The Brigham And Women's Hospital, Inc. Treatment of cancer
JP5697993B2 (ja) 2008-02-11 2015-04-08 アールエックスアイ ファーマシューティカルズ コーポレーション 修飾ＲＮＡｉポリヌクレオチドおよびその使用
US9217155B2 (en) 2008-05-28 2015-12-22 University Of Massachusetts Isolation of novel AAV'S and uses thereof
US8815818B2 (en) 2008-07-18 2014-08-26 Rxi Pharmaceuticals Corporation Phagocytic cell delivery of RNAI
CA2731779A1 (en) 2008-07-24 2010-01-28 Rxi Pharmaceuticals Corporation Rnai constructs and uses therof
AU2009273878A1 (en) 2008-07-25 2010-01-28 Alnylam Pharmaceuticals, Inc. Enhancement of siRNA silencing activity using universal bases or mismatches in the sense strand
CA2753338A1 (en) 2008-09-22 2010-03-25 Rxi Pharmaceuticals Corporation Neutral nanotransporters
ES2794401T3 (es) 2008-10-15 2020-11-18 Axolabs Gmbh Método de detección de oligonucleótidos
EP2358398A2 (en) 2008-10-24 2011-08-24 Isis Pharmaceuticals, Inc. Oligomeric compounds and methods
US9074211B2 (en) 2008-11-19 2015-07-07 Rxi Pharmaceuticals Corporation Inhibition of MAP4K4 through RNAI
WO2010078536A1 (en) 2009-01-05 2010-07-08 Rxi Pharmaceuticals Corporation Inhibition of pcsk9 through rnai
CA2750561C (en) 2009-01-26 2017-10-10 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein c-iii expression
US9745574B2 (en) 2009-02-04 2017-08-29 Rxi Pharmaceuticals Corporation RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
KR101141544B1 (ko) * 2009-03-13 2012-05-03 한국과학기술원 에스아이알엔에이 다중 접합체 및 이의 제조방법
US8309356B2 (en) 2009-04-01 2012-11-13 Yale University Pseudocomplementary oligonucleotides for targeted gene therapy
WO2010118263A1 (en) 2009-04-08 2010-10-14 University Of Massachusetts Single-nucleotide polymorphism (snp) targeting therapies for the treatment of huntington's disease
WO2010138263A2 (en) 2009-05-28 2010-12-02 University Of Massachusetts Novel aav 's and uses thereof
GB0910723D0 (en) 2009-06-22 2009-08-05 Sylentis Sau Novel drugs for inhibition of gene expression
PL2470656T3 (pl) 2009-08-27 2015-08-31 Idera Pharmaceuticals Inc Kompozycja do hamowania ekspresji genów i jej zastosowanie
US20150025122A1 (en) 2009-10-12 2015-01-22 Larry J. Smith Methods and Compositions for Modulating Gene Expression Using Oligonucleotide Based Drugs Administered in vivo or in vitro
EP2513337A4 (en) 2009-12-17 2013-12-25 Merck Sharp & Dohme METHOD FOR QUICKLY EVALUATING THE PERFORMANCE OF SHORT INTERFERING RNA WITH NEW CHEMICAL MODIFICATIONS
DK2534248T3 (en) 2010-02-08 2018-11-19 Ionis Pharmaceuticals Inc SELECTIVE REDUCTION OF ALLELVARIANS
WO2011109698A1 (en) 2010-03-04 2011-09-09 Rxi Pharmaceuticals Corporation Formulations and methods for targeted delivery to phagocyte cells
EP3578183B1 (en) 2010-03-24 2021-09-08 Phio Pharmaceuticals Corp. Rna interference in ocular indications
AU2011232365A1 (en) 2010-03-24 2012-10-25 Rxi Pharmaceuticals Corporation RNA interference in dermal and fibrotic indications
RU2615143C2 (ru) 2010-03-24 2017-04-04 Адвирна Самодоставляющие PHKi соединения уменьшенного размера
WO2011125943A1 (ja) 2010-04-01 2011-10-13 日本新薬株式会社 修飾オリゴヌクレオチド
WO2011139702A2 (en) 2010-04-28 2011-11-10 Isis Pharmaceuticals, Inc. Modified nucleosides and oligomeric compounds prepared therefrom
WO2012005898A2 (en) 2010-06-15 2012-01-12 Alnylam Pharmaceuticals, Inc. Chinese hamster ovary (cho) cell transcriptome, corresponding sirnas and uses thereof
EP2583692A4 (en) 2010-06-18 2014-11-26 Lsip Llc HEMMER OF THE EXPRESSION OF DOMINANT ALLELES
WO2012003330A2 (en) * 2010-06-30 2012-01-05 Stratos Genomics, Inc Multiplexed identification of nucleic acid sequences
WO2012083121A1 (en) 2010-12-16 2012-06-21 Vertex Pharmaceuticals Incorporated Inhibitors of influenza viruses replication
KR20130128435A (ko) 2010-12-16 2013-11-26 버텍스 파마슈티칼스 인코포레이티드 인플루엔자 바이러스 복제의 억제제
EP2651940A1 (en) 2010-12-16 2013-10-23 Vertex Pharmaceuticals Incorporated Inhibitors of influenza viruses replication
EP3192800A1 (en) 2010-12-17 2017-07-19 Arrowhead Pharmaceuticals, Inc. Galactose cluster-pharmacokinetic modulator targeting moiety for sirna
SG193280A1 (en) 2011-03-03 2013-10-30 Quark Pharmaceuticals Inc Oligonucleotide modulators of the toll-like receptor pathway
GB201105137D0 (en) 2011-03-28 2011-05-11 Isis Innovation Therapeutic molecules for use in the suppression of Parkinson's disease
WO2012143796A2 (en) 2011-04-21 2012-10-26 Institut Pasteur Korea Anti-inflammation compounds
AU2012272908A1 (en) 2011-06-21 2013-12-19 Alnylam Pharmaceuticals, Inc. Assays and methods for determining activity of a therapeutic agent in a subject
EP3358013B1 (en) 2012-05-02 2020-06-24 Sirna Therapeutics, Inc. Short interfering nucleic acid (sina) compositions
KR101567576B1 (ko) 2012-05-22 2015-11-10 올릭스 주식회사 세포 내 관통능을 가지고 ｒｎａ 간섭을 유도하는 핵산 분자 및 그 용도
KR102144140B1 (ko) 2012-07-10 2020-08-12 바제클리크 게엠베하 아난다미드-변형된 핵산 분자
US20150247141A1 (en) 2012-09-14 2015-09-03 Rana Therapeutics, Inc. Multimeric oligonucleotide compounds
WO2014047649A1 (en) 2012-09-24 2014-03-27 The Regents Of The University Of California Methods for arranging and packing nucleic acids for unusual resistance to nucleases and targeted delivery for gene therapy
RU2015119411A (ru) 2012-11-15 2017-01-10 Рош Инновейшен Сентер Копенгаген А/С Конъюгаты антисмысловых соединений, направленные на аполипопротеин в
CN104854242B (zh) 2012-12-05 2018-03-16 阿尔尼拉姆医药品有限公司 PCSK9 iRNA组合物及其使用方法
US20140200124A1 (en) 2013-01-15 2014-07-17 Karen Michelle JAMES Physical Therapy Device and Methods for Use Thereof
PL2992098T3 (pl) * 2013-05-01 2019-09-30 Ionis Pharmaceuticals, Inc. Kompozycje i sposoby modulowania ekspresji hbv i ttr
BR112015029276B1 (pt) 2013-05-22 2022-07-12 Alnylam Pharmaceuticals, Inc Agente de irna fita dupla capaz de inibir a expressão de tmprss6, composição farmacêutica e uso dos mesmos
JP7011389B2 (ja) 2013-06-12 2022-01-26 オンコイミューニン，インコーポレイティド オリゴヌクレオチドの全身性イン－ビボ送達
CA2915443A1 (en) 2013-06-16 2014-12-24 National University Corporation Tokyo Medical And Dental University Double-stranded antisense nucleic acid with exon-skipping effect
US9862350B2 (en) 2013-08-12 2018-01-09 Tk Holdings Inc. Dual chambered passenger airbag
GB201315141D0 (en) 2013-08-23 2013-10-09 Logicor R & D Ltd Improvements to electric heating systems and method of use thereof
DK3066219T3 (en) 2013-11-08 2019-03-11 Ionis Pharmaceuticals Inc METHODS FOR DETECTING OIGONUCLEOTIDES
EP3099795A4 (en) 2014-01-27 2018-01-17 The Board of Trustees of the Leland Stanford Junior University Oligonucleotides and methods for treatment of cardiomyopathy using rna interference
US10513710B2 (en) * 2014-04-18 2019-12-24 University Of Massachusetts Exosomal loading using hydrophobically modified oligonucleotides
MA39941A (fr) 2014-05-01 2017-03-08 Larry J Smith Procédés et modifications permettant de produire des composés arni simple brin à activité, potentiel et durée d'effet améliorés
EA201692370A1 (ru) 2014-05-22 2017-03-31 Элнилэм Фармасьютикалз, Инк. КОМПОЗИЦИИ иРНК АНГИОТЕНЗИНОГЕНА (AGT) И СПОСОБЫ ИХ ИСПОЛЬЗОВАНИЯ
US10588980B2 (en) 2014-06-23 2020-03-17 Novartis Ag Fatty acids and their use in conjugation to biomolecules
IL316808A (en) 2014-08-20 2025-01-01 Alnylam Pharmaceuticals Inc Modified double-stranded RNA materials and their uses
SG11201702877TA (en) 2014-10-10 2017-05-30 Hoffmann La Roche Galnac phosphoramidites, nucleic acid conjugates thereof and their use
JOP20200092A1 (ar) 2014-11-10 2017-06-16 Alnylam Pharmaceuticals Inc تركيبات iRNA لفيروس الكبد B (HBV) وطرق لاستخدامها
EP3271482A4 (en) 2015-03-17 2019-02-13 Arrowhead Pharmaceuticals, Inc. COMPOSITIONS AND METHODS FOR INHIBITING THE GENE EXPRESSION OF FACTOR XII
DK3277815T3 (da) 2015-04-03 2021-12-13 Univ Massachusetts Oligonukleotidforbindelser til behandling af præeklampsi og andre angiogene lidelser
JP6892433B2 (ja) 2015-04-03 2021-06-23 ユニバーシティ・オブ・マサチューセッツＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍａｓｓａｃｈｕｓｅｔｔｓ 十分に安定化された非対称ｓｉｒｎａ
DK3277814T3 (da) 2015-04-03 2020-08-24 Univ Massachusetts Oligonukleotidforbindelser til målretning mod huntingtin-mrna
JP6791875B2 (ja) 2015-04-10 2020-11-25 ハドソン アルファ インスティテュート フォー バイオテクノロジー ｍｉＲＮＡをブロックするための方法
IL316159A (en) 2015-06-15 2024-12-01 Mpeg La Llc Defined Oligonucleotide Multimers and Methods for Manufacture Thereof
MA43072A (fr) 2015-07-22 2018-05-30 Wave Life Sciences Ltd Compositions d'oligonucléotides et procédés associés
JP2018525001A (ja) 2015-08-06 2018-09-06 シティ・オブ・ホープＣｉｔｙ ｏｆ Ｈｏｐｅ 治療用細胞内在化コンジュゲート
US10633653B2 (en) 2015-08-14 2020-04-28 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
JP7511326B2 (ja) 2015-10-09 2024-07-05 ウェイブ ライフ サイエンシズ リミテッド オリゴヌクレオチド組成物およびその方法
CN105194689A (zh) 2015-10-10 2015-12-30 湖南中楚博生物科技有限公司 一种基于siRNA的双偶联复合物
JP7749201B6 (ja) 2016-01-31 2025-10-21 ユニバーシティー オブ マサチューセッツ 分岐オリゴヌクレオチド
MA45328A (fr) 2016-04-01 2019-02-06 Avidity Biosciences Llc Compositions acide nucléique-polypeptide et utilisations de celles-ci
WO2017174572A1 (en) 2016-04-04 2017-10-12 F. Hoffmann-La Roche Ag Nucleic acid sample preparation methods
US10036024B2 (en) 2016-06-03 2018-07-31 Purdue Research Foundation siRNA compositions that specifically downregulate expression of a variant of the PNPLA3 gene and methods of use thereof for treating a chronic liver disease or alcoholic liver disease (ALD)
WO2018031933A2 (en) 2016-08-12 2018-02-15 University Of Massachusetts Conjugated oligonucleotides
NZ751483A (en) 2016-09-01 2022-07-01 Proqr Therapeutics Ii Bv Chemically modified single-stranded rna-editing oligonucleotides
CN110520531A (zh) 2017-04-05 2019-11-29 赛伦斯治疗有限责任公司 产品和组合物
US11603532B2 (en) 2017-06-02 2023-03-14 Wave Life Sciences Ltd. Oligonucleotide compositions and methods of use thereof
US10844377B2 (en) 2017-06-23 2020-11-24 University Of Massachusetts Two-tailed self-delivering siRNA
CA3071086A1 (en) 2017-08-18 2019-02-21 The Procter & Gamble Company Cleaning agent
EP3550021A1 (en) 2018-04-05 2019-10-09 Silence Therapeutics GmbH Products and compositions for inhibiting expression of a target gene
KR20210093227A (ko) 2018-08-10 2021-07-27 유니버시티 오브 매사추세츠 Snp를 표적화하는 변형된 올리고뉴클레오티드
WO2020041769A1 (en) 2018-08-23 2020-02-27 University Of Massachusetts O-methyl rich fully stabilized oligonucleotides
WO2020150636A1 (en) 2019-01-18 2020-07-23 University Of Massachusetts Dynamic pharmacokinetic-modifying anchors
WO2020198509A2 (en) 2019-03-26 2020-10-01 University Of Massachusetts Modified oligonucleotides with increased stability
US20210115442A1 (en) 2019-08-23 2021-04-22 University Of Massachusetts O-methyl rich fully stabilized oligonucleotides
EP4090744A4 (en) 2020-01-17 2024-04-10 Anastasia Khvorova UNIVERSAL DYNAMIC PHARMACOKINETIC MODIFYING ANCHORS
AU2021244600A1 (en) 2020-03-26 2022-11-03 University Of Massachusetts Synthesis of modified oligonucleotides with increased stability
ES3014076T3 (en) 2020-03-26 2025-04-16 Nutrivert Inc Lipophilic enantiomers of desacetylglucosamine muramyl dipeptide with anti-inflammatory and growth promoting activity
US20210395739A1 (en) 2020-05-26 2021-12-23 University Of Massachusetts Synthetic oligonucleotides having regions of block and cluster modifications
EP4247391A4 (en) 2020-11-23 2025-11-19 Univ Massachusetts OLIGONUCLEOTIDES FOR DGAT2 MODULATION
IL309334A (en) 2021-06-23 2024-02-01 Univ Massachusetts Anti-FLT1 optimized oligonucleotide compounds for the treatment of preeclampsia and other angiogenic disorders

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6489464B1 (en) * 1994-06-01 2002-12-03 Hybridon, Inc. Branched oligonucleotides as pathogen-inhibitory agents
US20060009409A1 (en) * 2002-02-01 2006-01-12 Woolf Tod M Double-stranded oligonucleotides
US20070004664A1 (en) * 2002-09-05 2007-01-04 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALLERSON ET AL.: "Fully 2'-Modified Oligonucleotide Duplexes with Improved in Vitro Potency and Stability Compared to Unmodified Small Interfering RNA", JOURNAL OF MEDICINAL CHEMISTRY, vol. 48, no. 4, 20 January 2005 (2005-01-20), pages 901 - 904, XP002422086 *
PUBCHEM CID 12454428, 8 February 2007 (2007-02-08), pages 3, XP055401679, [retrieved on 20170413] *
PUBCHEM CID 22136768, 5 December 2007 (2007-12-05), pages 3, XP055401673, [retrieved on 20170413] *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12173286B2 (en) 2015-04-03 2024-12-24 University Of Massachusetts Fully stabilized asymmetric siRNA
US12077755B2 (en) 2015-08-14 2024-09-03 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides
US12049627B2 (en) 2017-06-23 2024-07-30 University Of Massachusetts Two-tailed self-delivering siRNA
WO2019183000A1 (en) * 2018-03-19 2019-09-26 University Of Massachusetts Modified guide rnas for crispr genome editing
EP3833763A4 (en) * 2018-08-10 2023-07-19 University of Massachusetts MODIFIED OLIGONUCLEOTIDES FOR TARGETING SNPS
US11827882B2 (en) 2018-08-10 2023-11-28 University Of Massachusetts Modified oligonucleotides targeting SNPs
US12297430B2 (en) 2018-08-23 2025-05-13 University Of Massachusetts O-methyl rich fully stabilized oligonucleotides
US12180477B2 (en) 2019-01-18 2024-12-31 University Of Massachusetts Dynamic pharmacokinetic-modifying anchors
CN113811311A (zh) * 2019-03-15 2021-12-17 马萨诸塞大学 用于组织特异性apoe调节的寡核苷酸
EP3937951A4 (en) * 2019-03-15 2023-04-05 University Of Massachusetts Oligonucleotides for tissue specific apoe modulation
EP3946374A4 (en) * 2019-03-29 2023-07-19 University of Massachusetts OLIGONUCLEOTIDE-BASED MODULATION OF C9ORF72
US12378554B2 (en) 2019-05-06 2025-08-05 University Of Massachusetts Anti-C9ORF72 oligonucleotides and related methods
JP2022547790A (ja) * 2019-08-09 2022-11-16 ユニバーシティー オブ マサチューセッツ Ｓｎｐを標的とする化学修飾オリゴヌクレオチド
US12024706B2 (en) 2019-08-09 2024-07-02 University Of Massachusetts Modified oligonucleotides targeting SNPs
US20210115442A1 (en) * 2019-08-23 2021-04-22 University Of Massachusetts O-methyl rich fully stabilized oligonucleotides
US12365894B2 (en) 2019-09-16 2025-07-22 University Of Massachusetts Branched lipid conjugates of siRNA for specific tissue delivery
US12258566B2 (en) 2020-02-28 2025-03-25 University Of Massachusetts Oligonucleotides for PRNP modulation
US20210363523A1 (en) * 2020-03-18 2021-11-25 University Of Massachusetts Oligonucleotides for mapt modulation
US12077758B2 (en) 2020-05-28 2024-09-03 University Of Massachusetts Oligonucleotides for SARS-CoV-2 modulation
US11702659B2 (en) 2021-06-23 2023-07-18 University Of Massachusetts Optimized anti-FLT1 oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders
EP4560019A1 (en) 2023-11-22 2025-05-28 Sylentis S.A.U. Sirna and compositions for prophylactic and therapeutic treatment of ocular retinal conditions
EP4560020A1 (en) 2023-11-22 2025-05-28 Sylentis S.A.U. Sirna and compositions for prophylactic and therapeutic treatment of ocular retinal conditions
WO2025109162A1 (en) 2023-11-22 2025-05-30 Sylentis S.A.U. Sirna and compositions for prophylactic and therapeutic treatment of ocular retinal conditions
WO2025109166A1 (en) 2023-11-22 2025-05-30 Sylentis S.A.U. Sirna and compositions for prophylactic and therapeutic treatment of ocular retinal conditions

Also Published As

Publication number Publication date
US20190247507A1 (en) 2019-08-15
US20210085793A1 (en) 2021-03-25
EP3408391A4 (en) 2019-08-28
AU2017210726A1 (en) 2018-08-16
CA3011894A1 (en) 2017-08-03
US20240293550A1 (en) 2024-09-05
JP2023040012A (ja) 2023-03-22
US10478503B2 (en) 2019-11-19
AU2017210726B2 (en) 2023-08-03
US11896669B2 (en) 2024-02-13
HK1258758A1 (zh) 2019-11-22
JP7749201B6 (ja) 2025-10-21
JP7749201B2 (ja) 2025-10-06
AU2023206109A1 (en) 2023-08-17
JP2019503394A (ja) 2019-02-07
JP2025066730A (ja) 2025-04-23
EP3408391A1 (en) 2018-12-05
US10799591B2 (en) 2020-10-13
US20170312367A1 (en) 2017-11-02

Similar Documents

Publication Publication Date Title
US20240293550A1 (en) Branched oligonucleotides
US12180477B2 (en) Dynamic pharmacokinetic-modifying anchors
US20220042015A1 (en) Conjugated oligonucleotides for tissue specific delivery
WO2021146548A1 (en) Universal dynamic pharmacokinetic-modifying anchors
WO2024129710A1 (en) Carbohydrate conjugates for the delivery of therapeutic oligonucleotides
WO2023224979A1 (en) Optimized sirna scaffolds
US20240191230A1 (en) Conjugates of sirna and antisense oligonucleotides (sirnaso) and methods of use in gene silencing
US20240360444A1 (en) Dendritic conjugates for the brain delivery of therapeutic oligonucleotides
WO2024186608A1 (en) Dendritic conjugates for the skin delivery of therapeutic oligonucleotides
WO2024196922A1 (en) Tumor delivery of dendritic oligonucleotides
WO2025085593A1 (en) Oligonucleotides with ethylene glycol modification

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17745083

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 3011894

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2018539903

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017210726

Country of ref document: AU

Date of ref document: 20170130

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2017745083

Country of ref document: EP