WO2023081500A2 - Conjugués oligonucleotides/arni - Google Patents

Conjugués oligonucleotides/arni Download PDF

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WO2023081500A2
WO2023081500A2 PCT/US2022/049230 US2022049230W WO2023081500A2 WO 2023081500 A2 WO2023081500 A2 WO 2023081500A2 US 2022049230 W US2022049230 W US 2022049230W WO 2023081500 A2 WO2023081500 A2 WO 2023081500A2
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Prior art keywords
nucleotides
oligonucleotide
sense strand
nucleotide
positions
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PCT/US2022/049230
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WO2023081500A3 (fr
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Bob Dale Brown
Maire JUNG
Travis GRIM
Matthew COSTALES
Marc Abrams
Martin Lee KOSER
Beata KAMINSKA
Jessica LAPIERRE
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Dicerna Pharmaceuticals, Inc.
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Publication of WO2023081500A2 publication Critical patent/WO2023081500A2/fr
Publication of WO2023081500A3 publication Critical patent/WO2023081500A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—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 against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01003—Aldehyde dehydrogenase (NAD+) (1.2.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the disclosure relates to oligonucleotides linked to lipid moieties useful in the inhibition of target genes in a variety of tissues.
  • the present disclosure relates to oligonucleotide-lipid conjugates, methods to prepare them, their chemical configuration, and methods to modulate (e.g., inhibit or reduce) the expression of a target gene using the conjugated nucleic acids and oligonucleotides according to the description provided herein.
  • the disclosure also provides pharmaceutically acceptable compositions comprising the conjugates of the present description and methods of using said compositions in the treatment of various diseases or disorders.
  • oligonucleotide or nucleic acid-based therapeutics have been under the clinical investigation, including antisense oligonucleotides (ASO), short interfering RNA (siRNA), double-stranded nucleic acids (dsNA), aptamers, ribozymes, exon-skipping and splice-altering oligonucleotides, immunomodulatory oligonucleotides, mRNAs, and CRISPR.
  • ASO antisense oligonucleotides
  • siRNA short interfering RNA
  • dsNA double-stranded nucleic acids
  • aptamers aptamers
  • ribozymes ribozymes
  • exon-skipping and splice-altering oligonucleotides immunomodulatory oligonucleotides
  • mRNAs mRNAs
  • CRISPR CRISPR
  • Dicer processed RNAi technologies utilize short double-stranded RNA (dsRNA) of approximately 21 base pair length with a two nucleotide (nt) 3 ’-overhang for the silencing of genes. These dsRNAs are generally called small interfering RNA (siRNA). siRNA 12 to 22 nucleotides in length are the active agent in RNAi. The siRNA duplex serves as a guide for mRNA degradation. Upon siRNA incorporation into the RNA-induced silencing complex (RISC) the complex interacts with a specific mRNA and ultimately suppresses the mRNA signal.
  • RISC RNA-induced silencing complex
  • the sense strand or passenger strand of siRNA is typically cleaved at the 9th nucleotide downstream from the 5 ’-end of the sense strand by Argonaute 2 (Ago2) endonuclease.
  • Ago2 Argonaute 2
  • the activated RISC complex containing the antisense strand or guide strand binds to the target mRNA through Watson-Crick base pairing causing degradation or translational blocking of the targeted RNA.
  • RNAi or siRNA molecules as pharmaceuticals has remained difficult due to obstacles encountered such as low biostability and unacceptable toxicity possibly caused by off-target effects.
  • Various types of chemical modifications to improve the pharmacokinetics and to overcome bio-instability problems have been investigated over the years to improve the stability and specificity of the RNAi duplexes.
  • the chemical modification in siRNAs has improved the serum stability of siRNAs.
  • RNAi activity was lost, but the careful placement of some specific modified residues enables enhanced siRNA biostability without loss of siRNA potency.
  • Some of these modifications have reduced siRNA side effects, such as the induction of recipient immune responses and inherent off-targeting effects and have even enhanced siRNA potency.
  • BNA bridged nucleic acids
  • LNA locked nucleic acid
  • RNAi oligonucleotide-based therapeutics comprising siRNAs or double-stranded nucleic acids (dsNAs) offer the potential for considerable expansion of the druggable target space and the possibility for treating orphan diseases that may be therapeutically unapproachable by other drug modalities (e.g., antibodies and/or small molecules).
  • RNAi oligonucleotide-based therapeutics that inhibit or reduce expression of specific target genes in the liver have been developed and are currently in clinical use (Sehgal et al., (2013) IOURNAL OF HEPATOLOGY 59: 1354-59).
  • RNAi oligonucleotides in extrahepatic cells, tissues, and organs.
  • RNAi triggers such as double stranded RNAs have become ubiquitous tools in biological research, and extensive basic and clinical development efforts have recently culminated in the FDA approval of ONPATTRO tm , the first RNAi drug.
  • ONPATTRO tm the first RNAi drug.
  • the difficulty of delivering RNAi agents to specific populations of disease related cells and or tissues, particularly outside the liver continues to limit the potential of RNAi therapy.
  • Repeated attempts over the past several years to develop useful, active, and persistent RNAi agents and structures for use based on known liver delivery technology have not convincingly demonstrated the intended effects outside the liver.
  • new dsRNA’s with variant structures have been developed to overcome the limitations in the field.
  • lipid-conjugated RNAi oligonucleotides that are capable of inhibiting expression of a target gene in hepatic and extrahepatic tissues.
  • lipid-conjugated RNAi oligonucleotides having a stem-loop at the 5’ end of the oligonucleotide showed comparable efficacy in reducing target gene expression in several regions of the central nervous system as a lipid-conjugated RNAi oligonucleotide having a stem-loop at the 3’ end of the oligonucleotide.
  • a stable stem-loop e.g., UACG
  • a relatively less stable stem-loop e.g., GAAA
  • the presence of a Tm- increasing nucleotide (e.g., locked nucleic acid) and/or truncation of a sense strand at the 3’ terminus improved reduction of target gene expression.
  • lipid-conjugated RNAi oligonucleotides having a “double-overhang” e.g., an overhang of at least one nucleotide at each of the 5’ and 3’ termini of an antisense strand
  • double-overhang e.g., an overhang of at least one nucleotide at each of the 5’ and 3’ termini of an antisense strand
  • target gene expression in the central nervous system at comparable levels relative to an RNAi oligonucleotide having only one overhang, i.e., at the 3 ’terminus of the antisense strand.
  • Up to three nucleotide truncations at the 3 ’terminus of the sense strand was tolerated, whereas introduction of a Tm-increasing nucleotide allowed for truncations of up to four nucleotides.
  • RNAi oligonucleotides delivered to the eye can effectively reduce expression of an ocular mRNA.
  • double-overhang RNAi oligonucleotides having a lipid conjugated to the 5’ terminal nucleotide of the sense strand reduced expression of a target gene in the optic nerve and retina.
  • RNAi oligonucleotides capable of reducing expression of a target gene in a macrophage of the liver.
  • RNAi oligonucleotides having a blunt end comprising the 3’ terminus of the sense strand and the 5 ’terminus of the antisense strand, and an overhang of up to seven nucleotides on the 3 ’terminus of the antisense strand, resulted in reduced expression of a gene expressed in macrophages.
  • lipid- conjugated RNAi oligonucleotides having a double-overhang, with or without Tm-increasing nucleotides similarly reduced expression of the macrophage target gene.
  • RNAi oligonucleotides are useful for targeting macrophages and treating liver diseases, including, but not limited to, drug or alcohol toxicity, steatosis, infection (e.g., viral infection), inflammatory liver diseases, fibrosis, hepatocellular carcinoma, and cirrhosis.
  • liver diseases including, but not limited to, drug or alcohol toxicity, steatosis, infection (e.g., viral infection), inflammatory liver diseases, fibrosis, hepatocellular carcinoma, and cirrhosis.
  • RNAi oligonucleotides modified by the addition of LNA, 2'-O-methyl modification, or phosphorothioate (PS) modification; either or both termini of the sense strand are modified with PS modification, 2'-O-methyl modification, or both; or the single strand overhang of the antisense sense strand is modified by LNA modification, 2'-O-methyl modification, PS modification, or any combination thereof.
  • the sense strand and the antisense strand of the RNAi trigger are modified by different chemical modifications.
  • lipid-conjugated RNAi oligonucleotides having either a blunt end or a stem-loop at the 3 ’terminus of the oligonucleotides, with truncated sense strands (e.g., having an overhang at the 3 ’terminus of the antisense strand) and Tm-increasing nucleotides, are capable of reducing target gene expression in several tissues, including the liver, skeletal muscle, adipose and adrenal.
  • lipid-conjugated RNAi oligonucleotides described herein are useful for reducing expression of a target gene in both hepatic and extrahepatic tissues.
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 8-20 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 8-20 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang of at least one nucleotide and a 3’ overhang of at least one nucleotide, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.
  • the 5’overhang is about 1-10 nucleotides. In some aspects, the 5’overhang is about 2-10 nucleotides. In some aspects, the 5’overhang is about 1-6 nucleotides. In some aspects, the 3’overhang is about 2-8 nucleotides. In some aspects, the 3’overhang is about 2-12 nucleotides. In some aspects, the 5’overhang is 2 nucleotides and the 3’overhang is about 3-7 nucleotides. In some aspects, the 3’overhang is 2 nucleotides, and the 5’overhang is about 2-8 nucleotides. In some aspects, the 3’overhang is 6-8 nucleotides, and the 5’overhang is about 2-4 nucleotides.
  • the sense strand is 18 nucleotides
  • the duplex region is 18 nucleotides
  • the 5’overhang is 2 nucleotides
  • the 3’overhang is 2 nucleotides
  • the sense strand is 17 nucleotides
  • the duplex region is 17 nucleotides
  • the 5’overhang is 3 nucleotides
  • the 3’overhang is 2 nucleotides
  • the sense strand is 16 nucleotides
  • the duplex region is 16 nucleotides
  • the 5’overhang is 4 nucleotides
  • the 3’overhang is 2 nucleotides
  • the sense strand is 13 nucleotides
  • the duplex region is 13 nucleotides
  • the 5’ overhang is 2 nucleotides
  • the 3’overhang is 7 nucleotides.
  • the sense strand is 12 nucleotides
  • the duplex region is 12 nucleotides
  • the 5’overhang is 2 nucleotides
  • the 3’overhang is 8 nucleotides
  • the sense strand is 12 nucleotides
  • the duplex region is 12 nucleotides
  • the 5’overhang is 3 nucleotides
  • the 3’overhang is 7 nucleotides
  • the sense strand is 10 nucleotides
  • the duplex region is 10 nucleotides
  • the 5’overhang is 1 nucleotide
  • the 3’overhang is 11 nucleotides.
  • the sense strand is 13 nucleotides, the duplex region is 13 nucleotides, the 5’ overhang is 2 nucleotides, and the 3’ overhang is 7 nucleotides.
  • the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 2 nucleotides, and the 3’overhang is 8 nucleotides.
  • the sense strand is 12 nucleotides, the duplex region is 12 nucleotides, the 5’overhang is 3 nucleotides, and the 3’overhang is 7 nucleotides.
  • the sense strand is 10 nucleotides, the duplex region is 10 nucleotides, the 5’overhang is 1 nucleotide, and the 3’overhang is 11 nucleotides.
  • the lipid moiety is selected from:
  • the lipid moiety is a hydrocarbon chain.
  • the hydrocarbon chain is a C8-C30 hydrocarbon chain.
  • the hydrocarbon chain is a C16 hydrocarbon chain.
  • the C16 hydrocarbon chain is represented by
  • the hydrocarbon chain is a C22 hydrocarbon chain. In some aspects, the C22 hydrocarbon chain is represented by
  • the lipid moiety is conjugated to the 5 ’terminal nucleotide of the sense strand. In some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.
  • the antisense strand is 22 nucleotides, wherein positions are numbered 1-22 from 5’ to 3’.
  • the nucleotide conjugated to the lipid moiety forms a base pair with a nucleotide at position 14 of the antisense strand, wherein positions are numbered 5’ to 3’.
  • the nucleotide conjugated to the lipid moiety forms a base pair with a nucleotide at position 12, 14, or 16 of the antisense strand, wherein positions are numbered 5’ to 3’.
  • the region of complementarity is fully complementary to the mRNA target sequence. In other aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some aspects, the region of complementarity comprises up to four mismatches to the mRNA target sequence.
  • the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a central nervous system (CNS) target sequence.
  • the CNS target sequence is a neuronal mRNA target sequence or an ocular mRNA target sequence.
  • the mRNA target sequence is a liver mRNA target sequence.
  • the liver mRNA target sequence is a liver macrophage mRNA target sequence.
  • the liver mRNA target sequence is a liver hepatocyte mRNA target sequence.
  • the liver mRNA target sequence is a liver sinusoidal endothelial cell mRNA target sequence.
  • the mRNA target sequence is an ocular mRNA target sequence.
  • the oligonucleotide comprises at least one modified nucleotide.
  • the modified nucleotide comprises a 2'-modification.
  • each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification.
  • the 2'-modification is a modification selected from 2'- aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-P-d- arabinonucleic acid.
  • the sense strand comprises nucleotide positions numbered 5’ to 3’, wherein each of positions 8-11 comprise a 2'-fluoro modification. In some aspects, the sense strand comprises a 2’ -fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10-13 of the antisense strand, wherein positions are numbered 5’ to 3’. In some aspects, the antisense strand comprises 22 nucleotides with positions 1-22 from 5' to 3', and wherein each of positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro modification. In some aspects, the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the oligonucleotide comprises at least one modified internucleotide linkage.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5’ to 3’.
  • the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 13 and 14, and between positions 14 and 15. In some aspects, the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.
  • the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • the phosphorylated nucleotide is uridine.
  • the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.
  • the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.
  • the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.
  • the sense strand comprises at least one Tm- increasing nucleotide. In some aspects, the sense strand comprises up to four Tm-increasing nucleotides. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm-increasing nucleotide is a locked nucleic acid.
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified intemucleotide linkage.
  • the sense strand comprises a 2’-fluoro modification at positions 8-11, numbered 5’ to 3’.
  • the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 16 and 17, and 17 and 18, numbered 5’ to 3’.
  • the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’.
  • the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine.
  • the lipid moiety is a C16 hydrocarbon represented by:
  • the lipid moiety is a C22 hydrocarbon represented by: some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the 5’overhang is 2 nucleotides and the 3’overhang is 2 nucleotides.
  • the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.
  • the mRNA target sequence is a liver mRNA target sequence, optionally a liver macrophage mRNA target sequence, a liver hepatocyte mRNA target sequence, or a liver sinusoidal endothelial cell mRNA target sequence.
  • the mRNA target sequence is an ocular mRNA target sequence.
  • the mRNA target sequence is an astrocyte mRNA target sequence.
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 8-11 and a 2’-O-methyl modification at positions 2-7 and 12-18, wherein the sense strand comprises a phosphorothioate linkage between positions 1
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to an ocular mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 8-11 and a 2’-O-methyl modification at positions 2-7 and 12-18, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 18 nucleotides in length, wherein the antisense and sense strands form a duplex region of 18 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to a macrophage mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’ -fluoro modification at positions 8-11 and a 2’-O- methyl modification at positions 2-7 and 12-18, wherein the sense strand comprises a phosphorothioate linkage between positions
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 17 nucleotides in length, wherein the antisense and sense strands form a duplex region of 17 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least three nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified intemucleotide linkage.
  • the sense strand comprises a 2’-fluoro modification at positions 8-11, numbered 5’ to 3’.
  • the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 15 and 16, and 16 and 17, numbered 5’ to 3’.
  • the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’.
  • the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine.
  • the lipid moiety is a C16 hydrocarbon represented by:
  • the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.
  • the 5’overhang is 3 nucleotides and the 3’overhang is 2 nucleotides.
  • the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence.
  • CNS central nervous system
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 17 nucleotides in length, wherein the antisense and sense strands form a duplex region of 17 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising three nucleotides and a 3’ overhang comprising two nucleotides, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a 2’-fluoro modification at positions 8-11 and a 2-O-methyl modification at positions 2-7 and 12-17, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, positions 15 and
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 16 nucleotides in length, wherein the antisense and sense strands form a duplex region of 16 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least four nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises up to five Tm-increasing nucleotides, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified intern
  • the sense strand comprises a 2’-fluoro modification at positions 8-11, numbered 5’ to 3’.
  • the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 14 and 15, and 15 and 16, numbered 5’ to 3’.
  • the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’.
  • the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine.
  • the lipid moiety is a C16 hydrocarbon represented by:
  • the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.
  • the 5’overhang is 4 nucleotides and the 3’overhang is 2 nucleotides.
  • the sense strand comprises 1-5, 1-4, 1-3, or 1-2 Tm-increasing nucleotides. In some aspects, the sense strand comprises 1, 2, 3, 4 or 5 Tm-increasing nucleotides. In some aspects, the sense strand comprises up to three Tm-increasing nucleotides.
  • the sense strand comprises a Tm-increasing nucleotide at positions 2, 15 and 16, numbered 5’ to 3’.
  • the Tm-increasing nucleotide is a bicyclic nucleotide, optionally, a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • the region of complementarity is fully complementary to the mRNA target sequence.
  • the region of complementarity is partially complementary to the mRNA target sequence.
  • the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.
  • CNS central nervous system
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 16 nucleotides in length, wherein the antisense and sense strands form a duplex region of 16 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least four nucleotides and a 3’ overhang comprising at least two nucleotides, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5’ terminal nucleotide of the sense strand, wherein the sense strand comprises a Tm-increasing nucleotide at positions 2, 15 and 16, wherein the sense strand comprises a 2’-fluoro modification at positions 8-11 and a 2-O-methyl modification
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 13 nucleotides in length, wherein the antisense and sense strands form a duplex region of 13 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least seven nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to an internal nucleotide of the sense strand, wherein the sense strand comprises up to three Tm-increasing nucleotides, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucle
  • the sense strand comprises a 2’-fluoro modification at positions 3-6, numbered 5’ to 3’.
  • the antisense strand comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 11 and 12, and 12 and 13, numbered 5’ to 3’.
  • the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’.
  • the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine.
  • the lipid moiety is conjugated at a nucleotide at position 2 of the sense strand, numbered 5’ to 3’.
  • the lipid moiety is a C22 hydrocarbon represented by: some aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.
  • the 5’overhang is 2 nucleotides and the 3’overhang is 7 nucleotides.
  • the sense strand comprises 1-3, or 1-2 Tm-increasing nucleotides. In some aspects, the sense strand comprises 1, 2, or 3 Tm-increasing nucleotides.
  • the sense strand comprises a Tm-increasing nucleotide at (i) positions 1 and 10, or (ii) positions 1, 10 and 11, numbered 5’ to 3’.
  • the Tm-increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • the region of complementarity is fully complementary to the mRNA target sequence.
  • the region of complementarity is partially complementary to the mRNA target sequence.
  • the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a liver target sequence.
  • the mRNA target sequence is a hepatocyte target sequence. In some aspects, the mRNA target sequence is a liver sinusoidal endothelial cell mRNA target sequence. In some aspects, the mRNA target sequence is a macrophage mRNA target sequence, optionally a liver macrophage mRNA target sequence.
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 13 nucleotides in length, wherein the antisense and sense strands form a duplex region of 13 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising seven nucleotides, wherein the antisense strand comprises a region of complementarity to a macrophage mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide at position 2 of the sense strand, wherein the sense strand comprises a Tm-increasing nucleotide at positions 1, and 11, wherein the sense strand comprises a 2’-fluoro modification at positions 3-6 and a 2-0- methyl modification at positions 7
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 13 nucleotides in length, wherein the antisense and sense strands form a duplex region of 13 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising two nucleotides and a 3’ overhang comprising seven nucleotides, wherein the antisense strand comprises a region of complementarity to a macrophage mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide at position 2 of the sense strand, wherein the sense strand comprises a Tm-increasing nucleotide at positions 1, 10, and 11, wherein the sense strand comprises a 2’-fluoro modification at positions 3-6 and a 2-O- methyl modification at positions 7
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 12 nucleotides in length, wherein the antisense and sense strands form a duplex region of 12 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising at least two nucleotides and a 3’ overhang comprising at least seven nucleotides, wherein the antisense strand comprises a region of complementarity to an mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5 ’terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.
  • the sense strand comprises a 2’-fluoro modification at positions 3-6 or 4-7, numbered 5’ to 3’.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 10 and 11, and 11 and 12, numbered 5’ to 3’.
  • the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the 5’overhang is 2 nucleotides and the 3 ’overhang is 8 nucleotides. In some aspects, the 5’overhang is 3 nucleotides and the 3 ’overhang is 7 nucleotides.
  • the oligonucleotide comprises (i) 1-3 or 1-2 Tm-increasing nucleotides, or (ii) 1, 2 or 3 Tm-increasing nucleotides.
  • the sense strand comprises a Tm-increasing nucleotide at (i) positions 2, 10 and 11, or (ii) positions 2, 11 and 12, numbered 5’ to 3’. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 10 nucleotides in length, wherein the antisense and sense strands form a duplex region of 10 base pairs, wherein the antisense strand comprises an orientation of 5’ to 3’, wherein the antisense strand comprises a 5’ overhang comprising one nucleotide and a 3’ overhang comprising eleven nucleotides, wherein the antisense strand comprises a region of complementarity to an mRNA target sequence, wherein the sense strand comprises at least one lipid moiety conjugated to a 5 ’terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprise at least one 2’ -modified nucleotide and at least one modified internucleotide linkage.
  • the at least one modified intemucleotide linkage is a phosphorothioate linkage.
  • the sense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10, numbered 5’ to 3’.
  • the antisense strand comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the oligonucleotide comprises (i) 1-3 Tm- increasing nucleotides, or (ii) 1, 2 or 3 Tm-increasing nucleotides.
  • the sense strand comprises a Tm-increasing nucleotide at positions 2, 6 and 7.
  • the Tm- increasing nucleotide is a bicyclic nucleotide, optionally a locked nucleic acid (LNA).
  • the antisense strand comprises a 2’ -fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification
  • the oligonucleotide is a Dicer substrate. In some aspects, the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and/or in vivo.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an oligonucleotide described herein, and a pharmaceutically acceptable carrier, delivery agent or excipient.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.
  • the target mRNA is expressed in the central nervous system, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and/or lumbar spinal cord.
  • the target mRNA is expressed in a neuron of the central nervous system.
  • the target mRNA is expressed in a macrophage.
  • the macrophage is in the liver.
  • the target mRNA is expressed in the liver. In some aspects, the target mRNA is expressed in a hepatocyte. In some aspects, the target mRNA is expressed in a liver sinusoidal endothelial cell. In some aspects, the target mRNA is expressed in ocular tissue. In some aspects, the target mRNA is expressed in a tissue of the central nervous system, liver tissue, ocular tissue, adipose tissue, muscle tissue, adrenal tissue, cardiac tissue, lung tissue, or any combination thereof.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an mRNA of the central nervous system, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and/or lumbar spinal cord.
  • the mRNA of the central nervous system is a neuronal mRNA.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an mRNA of the liver, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an ocular mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a macrophage mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.
  • the macrophage mRNA is expressed in the liver.
  • the disclosure provides a method of delivering an oligonucleotide to a cell or population of cells in the central nervous system, liver tissue or ocular tissue, the method comprising administering a pharmaceutical composition described herein.
  • the disclosure provides a method of reducing expression of a target mRNA in a subject, comprising administering to the subject an oligonucleotide or pharmaceutical composition described herein.
  • the target mRNA is expressed in the central nervous system, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and/or lumbar spinal cord.
  • the target mRNA is expressed in a neuron of the central nervous system.
  • the target mRNA is expressed in the liver.
  • the target mRNA is expressed in a hepatocyte.
  • the target mRNA is expressed in a liver sinusoidal endothelial cell. In some aspects, the target mRNA is expressed in a macrophage. In some aspects, the macrophage is in the liver. In some aspects, the target mRNA is expressed in ocular tissue. In other aspects, the disclosure provides a kit comprising an oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with the overexpression of a target mRNA.
  • the disclosure provides a kit comprising an oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with the reduction in expression of a target mRNA.
  • the disclosure provides use of an oligonucleotide or pharmaceutical composition described herein, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with the reduction in the expression of a target mRNA.
  • the disclosure provides use of an oligonucleotide or pharmaceutical composition described herein, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with the overexpression of a target mRNA.
  • the disclosure provides an oligonucleotide or pharmaceutical composition described herein, for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with expression of a target mRNA.
  • the target mRNA is expressed in the central nervous system, a neuron of the central system, the liver, a macrophage, optionally a macrophage in the liver, ocular tissue, or any combination thereof, optionally wherein the central nervous system comprises the frontal cortex, hippocampus, medulla, cerebellum, lumbar dorsal root ganglion, and/or lumbar spinal cord.
  • the disclosure provides a method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism an oligonucleotide described herein, said oligonucleotide being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism.
  • RNAi target-specific RNA interference
  • the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder.
  • the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 15-30 nucleotides in length and a sense strand of about 15-50 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 15-30 base pairs, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises (i) at least one lipid moiety conjugated to a nucleotide of the sense strand, and (ii) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2, and wherein the sense and antisense strands each comprise an orientation of 5’ to 3’, and wherein the stem-loop is at the 5’ end of the sense strand.
  • the oligonucleotide comprises a blunt end.
  • the blunt end comprises the 3’ end of the sense strand and the 5’ end of the antisense strand.
  • the oligonucleotide comprises an overhang of at least two nucleotides. In some aspects, the overhang comprises the 5 ’end of the antisense strand.
  • the sense strand is about 28-38 nucleotides. In some aspects, the antisense strand is 22 nucleotides. In some aspects, the lipid moiety is conjugated to a nucleotide comprising the loop.
  • the sense strand is 28 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 4, positions numbered 5’ to 3’;
  • the sense strand is 30 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 4, positions numbered 5’ to 3’;
  • the sense strand is 34 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 6 or position 15, positions numbered 5’ to 3’;
  • the sense strand is 38 nucleotides, and the lipid moiety is conjugated to a nucleotide at position 8, positions numbered 5’ to 3’.
  • the lipid moiety is selected from:
  • the lipid moiety is a hydrocarbon chain. In some aspects, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some aspects, the hydrocarbon chain is a C16 hydrocarbon chain. In some aspects, the C16 hydrocarbon chain is represented by In any of the foregoing or related aspects, the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.
  • the region of complementarity is fully complementary to the mRNA target sequence. In other aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence. In some aspects, the mRNA target sequence is an ocular mRNA target sequence.
  • CNS central nervous system
  • the loop sequence is 5’-GAAA-3’. In some aspects, the loop sequence is 5’-UNCG-3’, wherein N is any nucleotide. In some aspects, the loop sequence is 5’-UACG-3’.
  • the oligonucleotide comprises at least one modified nucleotide.
  • the modified nucleotide comprises a 2'-modification.
  • each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification.
  • the 2'-modification is a modification selected from 2'- aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-P-d- arabinonucleic acid.
  • the antisense strand comprises 22 nucleotides with positions 1-22 from 5' to 3', and wherein each of positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro modification. In some aspects,
  • the sense strand is 38 nucleotides with positions 1-38 from 5’ to 3’, and wherein each of positions 26-29 comprise a 2’ -fluoro modification;
  • the sense strand is 34 nucleotides with positions 1-34 from 5’ to 3’, and wherein each of positions 22-25 comprise a 2’ -fluoro modification;
  • the sense strand is 30 nucleotides with positions 1-30 from 5’ to 3’, and wherein each of positions 18-21 comprise a 2’ -fluoro modification;
  • the sense strand is 28 nucleotides with positions 1-28 from 5’ to 3’, and wherein each of positions 18-21 comprise a 2’ -fluoro modification.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5’ to 3’.
  • the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5' to 3'.
  • the sense strand comprises a phosphorothioate linkage between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.
  • the sense strand is 38 nucleotides with positions 1-38 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 36 and 37, and 37 and 38;
  • the sense strand is 34 nucleotides with positions 1-34 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 32 and 33, and 33 and 34;
  • the sense strand is 30 nucleotides with positions 1-30 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 28 and 29, and 29 and 30; or
  • the sense strand is 28 nucleotides with positions 1-28 from 5’ to 3’, and wherein the sense strand comprises a phosphorothioate linkage between positions 26 and 27, and 27 and 28.
  • the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • the phosphorylated nucleotide is uridine.
  • the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.
  • the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.
  • the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.
  • the sense strand comprises at least one Tm- increasing nucleotide. In some aspects, the sense strand comprises 1-6, 1-5, 1-4, 1-3 or 12 Tm-increasing nucleotides. In some aspects, the sense strand comprises 1, 2, 3, 4, 5 or 6 Tm- increasing nucleotides. In some aspects, the sense strand comprises up to six Tm-increasing nucleotides. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm-increasing nucleotide is a locked nucleic acid.
  • SI and S2 each comprise 1-6 nucleotides. In some aspects, SI and S2 each comprise 4 nucleotides. In some aspects, SI and S2 each comprise 2 nucleotides. In some aspects, SI and S2 each comprise at least one Tm-increasing nucleotide. In some aspects, SI and S2 are each 4 nucleotides, wherein 1-3 nucleotides of each SI and S2 are Tm-increasing nucleotides.
  • the remaining nucleotides comprise a 2'-O- methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the disclosure provides, a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 32-34 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 20-22 base pairs and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense and antisense strands each comprise an orientation of 5’ to 3’, wherein the stem-loop is at the 5’
  • the sense strand is 34 nucleotides and comprises a 2’ -fluoro modification at positions 22-25, numbered 5’ to 3’.
  • the antisense strand is 22 nucleotides and comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the sense strand is 34 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 32 and 33, and 33 and 34, numbered 5’ to 3’.
  • the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’.
  • the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine.
  • the lipid moiety is a C16 hydrocarbon represented by:
  • the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the lipid moiety is conjugated to a nucleotide at position 6 of the sense strand, numbered 5’ to 3’.
  • SI and S2 each comprise 1-6 nucleotides. In some aspects, SI and S2 are each 4 nucleotides. In some aspects, L is 4 nucleotides. In some aspects, L comprises the sequence 5’-GAAA-3’.
  • the region of complementarity is fully complementary to the mRNA target sequence. In other aspects, wherein the region of complementarity is partially complementary to the mRNA target sequence.
  • the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.
  • CNS central nervous system
  • the disclosure provides, a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 34 nucleotides in length, wherein the antisense and sense strands form a duplex region of 22 base pairs and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, wherein SI and S2 each comprise 4 nucleotides, wherein L forms a loop between SI and S2, and wherein L comprises four nucleotides, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense and antisense strands each
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 26-28 nucleotides in length, wherein the antisense and sense strands form an asymmetric duplex region of about 20-22 base pairs comprising a 3’ terminal overhang of at least 2 nucleotides of the antisense strand, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises: (i) a stemloop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’- S1-L-S2-3’, wherein SI is complementary to S2, wherein L forms a loop between SI and S2 and comprises the sequence UNCG, and wherein SI and S2 each comprise at least one Tm- increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucle
  • the sense strand is 28 nucleotides and comprises a 2’ -fluoro modification at positions 18-21, numbered 5’ to 3’.
  • the antisense strand is 22 nucleotides and comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3 ’.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the sense strand is 28 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 26 and 27, and 27 and 28, numbered 5’ to 3’.
  • the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the 3 ’terminal overhang is 2 nucleotides.
  • the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’.
  • the phosphorylated uridine is 4’-O- monomethylphosphonae-2’-O-methyl uridine.
  • the lipid moiety is a C16 hydrocarbon represented by:
  • the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the lipid moiety is conjugated to a nucleotide at position 4 of the sense strand, numbered 5’ to 3’.
  • SI and S2 each comprise 1-6 nucleotides. In some aspects, wherein SI and S2 are each 2 nucleotides. In some aspects, SI and S2 each comprise one Tm-increasing nucleotide. In some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide.
  • Tm-increasing nucleotide is a locked nucleic acid (LNA).
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • L is 4 nucleotides.
  • L comprises the sequence 5’-UNCG-3’, wherein N is any nucleotide.
  • L comprises the sequence 5’- UACG-3.
  • the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence.
  • the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a central nervous system (CNS) target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence.
  • CNS central nervous system
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 28 nucleotides in length, wherein the antisense and sense strands form an asymmetric duplex region of 22 base pairs comprising a 3’ terminal overhang of 2 nucleotides of the antisense strand, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises: (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L- S2-3’, wherein SI is complementary to S2, wherein SI and S2 each comprise 2 nucleotides, wherein L forms a loop between SI and S2 and comprises the sequence UNCG, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii)
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 20-22 nucleotides in length and a sense strand of about 32-34 nucleotides in length, wherein the antisense and sense strands form a duplex region of about 20-22 base pairs, and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, wherein the sense strand comprises: (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, wherein L forms a loop between SI and S2, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii) at least one lipid moiety conjugated to a nucleotide of the loop, wherein the sense strand comprises an
  • the sense strand is 34 nucleotides and comprises a 2’ -fluoro modification at positions 22-25, numbered 5’ to 3’.
  • antisense strand is 22 nucleotides and comprises a 2’-fluoro modification at positions 2-5, 7, 10 and 14, numbered 5’ to 3’.
  • the at least one modified intemucleotide linkage is a phosphorothioate linkage.
  • the sense strand is 34 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 32 and 33, and 33 and 34, numbered 5’ to 3’.
  • the antisense strand is 22 nucleotides and comprises phosphorothioate linkages between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22, numbered 5’ to 3’.
  • the oligonucleotide comprises a blunt end comprising the 5’ end of the antisense strand and the 3’ end of the sense strand.
  • the antisense strand comprises a phosphorylated uridine at position 1, numbered 5’ to 3’.
  • the phosphorylated uridine is 4’-O-monomethylphosphonae-2’-O-methyl uridine.
  • the lipid moiety is a C16 hydrocarbon represented by:
  • the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide. In some aspects, the lipid moiety is conjugated to a nucleotide at position 6 of the sense strand, numbered 5’ to 3’.
  • SI and S2 each comprise 1-6 nucleotides. In some aspects, SI and S2 are each 4 nucleotides. In some aspects, SI and S2 each comprise one Tm-increasing nucleotide, some aspects, the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm- increasing nucleotide is a locked nucleic acid (LNA).
  • LNA locked nucleic acid
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • L is 4 nucleotides. In some aspects, L comprises the sequence 5’-GAAA-3’.
  • the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is (i) a central nervous system (CNS) mRNA target sequence, optionally a neuronal mRNA target sequence or an ocular mRNA target sequence; or (ii) an ocular tissue mRNA target sequence.
  • CNS central nervous system
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length and a sense strand of 34 nucleotides in length, wherein the antisense and sense strands form a duplex region of 22 base pairs, and the oligonucleotide is blunt ended, wherein the antisense strand comprises a region of complementarity to a central nervous system mRNA target sequence, wherein the sense strand comprises: (i) a stem-loop, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, wherein SI and S2 each comprise 4 nucleotides, wherein L forms a loop between SI and S2, wherein the loop comprises 4 nucleotides, and wherein SI and S2 each comprise at least one Tm-increasing nucleotide, and (ii) at least one lipid mo
  • the oligonucleotide is a Dicer substrate. In further aspects, the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and/or in vivo.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an oligonucleotide of any of the foregoing or related aspects, and a pharmaceutically acceptable carrier, delivery agent or excipient.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.
  • the target mRNA is expressed in the central nervous system, optionally a neuron of the central nervous system.
  • the target mRNA is expressed in ocular tissue, optionally the optic nerve and/or retina.
  • the disclosure provides a method of delivering an oligonucleotide to a cell or population of cells in the central nervous system or ocular tissue, the method comprising administering a pharmaceutical composition described herein.
  • the disclosure provides a method of reducing expression of a target mRNA in a subject, comprising administering to the subject an oligonucleotide or pharmaceutical composition described herein.
  • target mRNA is expressed in the central nervous system, optionally a neuron of the central nervous system. In some aspects of the methods described herein, the target mRNA is expressed in ocular tissue, optionally the optic nerve and/or retina.
  • the disclosure provides a kit comprising an oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with expression of a target mRNA.
  • the disclosure provides use of an oligonucleotide or pharmaceutical composition described herein, in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with expression of a target mRNA.
  • the disclosure provides any oligonucleotide or pharmaceutical composition described herein, for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with expression of a target mRNA.
  • the target mRNA is expressed in the central nervous system and/or a neuron of the central nervous system. In some aspects, the target mRNA is expressed in ocular tissue, optionally the retina and/or optic nerve.
  • the central nervous system comprises the frontal cortex, the hippocampus, the cerebellum, the brainstem, lumbar dorsal root ganglion, the lumbar spinal cord, or combinations thereof.
  • the central nervous system comprises the frontal cortex, the hippocampus, the cerebellum, the brainstem, lumbar dorsal root ganglion, the lumbar spinal cord, or combinations thereof.
  • the disclosure provides a method of activating target-specific RNA interference (RNAi) in an organism comprising administering to said organism a dsRNA oligonucleotide described herein, said oligonucleotide being administered in an amount sufficient for degradation of the target mRNA to occur, thereby activating target-specific RNAi in the organism.
  • the target mRNA specifies the amino acid sequence of a protein involved or predicted to be involved in a human disease or disorder.
  • the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 13-30 nucleotides in length and a sense strand of about 10-50 nucleotides in length, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of about 2-10 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 10- 30 nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.
  • the disclosure provides a double-stranded oligonucleotide comprising an antisense strand of about 13-30 nucleotides in length and a sense strand of about 10-50 nucleotides in length, wherein the antisense and sense strands are separate strands which form an asymmetric duplex region having an overhang of about 2-12 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 10- 30 nucleotides, wherein the antisense strand comprises a region of complementarity to a mRNA target sequence, and wherein the sense strand comprises at least one lipid moiety conjugated to a nucleotide on the sense strand.
  • the oligonucleotide comprises a blunt end.
  • the blunt end comprises the 3’ end of the sense strand and the 5’ end of the antisense strand.
  • the oligonucleotide comprises a stem loop, wherein the stem loop comprises a nucleotide sequence represented by the formula: 5’-Sl-L-S2-3’, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2, and wherein the sense strand comprises an orientation of 5’ to 3’, and wherein the stem-loop is at the 3’ end of the sense strand.
  • the sense strand is 19 nucleotides
  • the duplex region is 19 nucleotides
  • the overhang is 3 nucleotides
  • the sense strand is 18 nucleotides
  • the duplex region is 18 nucleotides
  • the overhang is 4 nucleotides
  • the sense strand is 17 nucleotides
  • the duplex region is 17 nucleotides
  • the overhang is 5 nucleotides
  • the sense strand is 16 nucleotides
  • the duplex region is 16 nucleotides
  • the overhang is 6 nucleotides
  • the sense strand is 15 nucleotides
  • the duplex region is 15 nucleotides
  • the overhang is 7 nucleotides.
  • the sense strand is 19 nucleotides
  • the duplex region is 19 nucleotides
  • the overhang is 3 nucleotides
  • the sense strand is 18 nucleotides
  • the duplex region is 18 nucleotides
  • the overhang is 4 nucleotides
  • the sense strand is 17 nucleotides
  • the duplex region is 17 nucleotides
  • the overhang is 5 nucleotides
  • the sense strand is 16 nucleotides
  • the duplex region is 16 nucleotides
  • the overhang is 6 nucleotides
  • the sense strand is 15 nucleotides
  • the duplex region is 15 nucleotides
  • the overhang is 7 nucleotides
  • the sense strand is 14 nucleotides
  • the duplex region is 14 nucleotides
  • the overhang is 8 nucleotides
  • the sense strand is 13 nucleotides
  • the duplex region is 13 nucleotides
  • the overhang is 8 nucleotides
  • the sense strand is 12 nucleotides
  • the duplex region is 12 nucleotides
  • the overhang is 10 nucleotides.
  • the sense strand is 10 nucleotides
  • the duplex region is 10 nucleotides
  • the overhang is 12 nucleotides.
  • the sense strand is 32 nucleotides, the duplex region is 16 nucleotides, and the overhang is 6 nucleotides;
  • the sense strand is 30 nucleotides, the duplex region is 14 nucleotides, and the overhang is 8 nucleotides; or (iii) the sense strand is 28 nucleotides, the duplex region is 12 nucleotides, and the overhang is 10 nucleotides.
  • the antisense strand is 22 nucleotides, with positions 1-22 numbered 5’ to 3’.
  • the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 14 of the antisense strand. In some aspects, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 12 of the antisense strand. In some aspects, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 20, position 19, position 18, position 17, position 16, position 15, position 14, position 13, or position 12 of the antisense strand.
  • the lipid moiety is conjugated to a nucleotide in the loop. In some aspects, the lipid moiety is conjugated to the 3 ’terminal nucleotide on the sense strand. In some aspects, the lipid moiety is conjugated to the 5’ terminal nucleotide on the sense strand.
  • the lipid moiety is selected from:
  • the lipid moiety is a hydrocarbon chain.
  • the hydrocarbon chain is a C8-C30 hydrocarbon chain.
  • the hydrocarbon chain is a C16 hydrocarbon chain.
  • the C16 hydrocarbon chain is represented by
  • the hydrocarbon chain is a C22 hydrocarbon chain. In some aspects, the C22 hydrocarbon chain is represented by
  • the lipid moiety is conjugated to the 2’ carbon of the ribose ring of the nucleotide.
  • the region of complementarity is fully complementary to the mRNA target sequence. In some aspects, the region of complementarity is partially complementary to the mRNA target sequence. In some aspects, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the mRNA target sequence is a liver mRNA target sequence, optionally a liver macrophage mRNA target sequence, a liver hepatocyte mRNA target sequence, or a liver sinusoidal endothelial cell mRNA target sequence.
  • the mRNA target sequence is an ocular mRNA target sequence.
  • the mRNA target sequence is expressed in liver tissue, skeletal muscle tissue, adipose tissue and/or adrenal tissue.
  • the mRNA target sequence is expressed in at least one tissue of the central nervous system.
  • the at least one tissue of the central nervous system is selected from frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combination thereof.
  • oligonucleotide comprises at least one modified nucleotide.
  • the modified nucleotide comprises a 2'-modification.
  • each of the nucleotides of the sense strand and the antisense strand comprise a 2'-modification.
  • the 2'-modification is a modification selected from 2'- aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-P-d- arabinonucleic acid.
  • the antisense strand is 22 nucleotides with positions 1- 22 numbered 5’ to 3’, and wherein the sense strand comprises a 2’-fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10-13 of the antisense strand. In some aspects, the antisense strand is 22 nucleotides with positions 1-22 numbered 5’ to 3’, and wherein the sense strand comprises a 2’-fluoro modification at each of nucleotides forming a base pair with nucleotides at positions 10, 11, 12, 13, or any combination thereof, of the antisense strand.
  • the antisense strand comprises 22 nucleotides with positions 1-22 from 5' to 3', and wherein each of positions 2, 3, 4, 5, 7, 10 and 14 comprise a 2'-fluoro modification.
  • the remaining nucleotides comprise a 2'-O-methyl modification, provided that the nucleotide of the sense strand conjugated to the at least one lipid moiety does not comprise a 2’-O-methyl modification.
  • the oligonucleotide comprises at least one modified internucleotide linkage.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the antisense strand comprises a phosphorothioate linkage (i) between positions 1 and 2, and between positions 2 and 3; or (ii) between positions 1 and 2, between positions 2 and 3, and between positions 3 and 4, wherein positions are numbered 1-4 from 5’ to 3’.
  • the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a phosphorothioate linkage between positions 20 and 21 and between positions 21 and 22, wherein positions are numbered 1-22 from 5' to 3'. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20.
  • the antisense strand comprises a phosphorothioate linkage between positions 15 and 16, between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20. In some aspects, the antisense strand comprises a phosphorothioate linkage between positions 12 and 13, between positions 15 and 16, between positions 16 and 17, between positions 17 and 18, between positions 18 and 19, and between positions 19 and 20. In some aspects, the sense strand comprises a phosphorothioate linkage between positions 1 and 2.
  • the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between the penultimate nucleotide and third nucleotide from the 3’ end, and between the penultimate nucleotide and ultimate nucleotide.
  • the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • the phosphorylated nucleotide is uridine.
  • the 4'-carbon of the sugar of the 5 '-nucleotide of the antisense strand comprises a phosphate analog.
  • the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.
  • the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.
  • the sense strand comprises at least one Tm- increasing nucleotide. In some aspects, the sense strand comprises up to nine Tm-increasing nucleotides. In some aspects, the sense strand comprises 1-3 Tm-increasing nucleotides. In some aspects, SI and S2 each comprise at least one Tm-increasing nucleotide. In some aspects, SI and S2 are each 3 nucleotides, and each nucleotide is a Tm-increasing nucleotide.
  • the sense strand comprises up to three Tm-increasing nucleotides at nucleotide positions, provided the nucleotide positions are not in the stem-loop. In some aspects, wherein the sense strand comprises up to five Tm-increasing nucleotides.
  • the Tm-increasing nucleotide is a bicyclic nucleotide. In some aspects, the Tm-increasing nucleotide is a locked nucleic acid. In some aspects, the sense strand is 20 nucleotides in length, wherein the nucleotides are numbered 1- 20 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:
  • the sense strand is 16 nucleotides in length, wherein the nucleotides are numbered 1-16 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:
  • the sense strand is 14 nucleotides in length, wherein the nucleotides are numbered 1-14 5’ to 3, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:
  • the sense strand is 12 nucleotides in length, wherein the nucleotides are numbered 1-12 5’ to 3’, and wherein the sense strand comprises a locked nucleic acid at a nucleotide located at:
  • the sense strand comprises a locked nucleic acid at a nucleotide that forms a base pair with a nucleotide at a position of the antisense strand selected from:
  • the oligonucleotide is a Dicer substrate. In some aspects, the oligonucleotide reduces expression of the mRNA target sequence in a cell or population of cells in vitro and/or in vivo. In some aspects, the disclosure provides a pharmaceutical composition comprising any oligonucleotide described herein, and a pharmaceutically acceptable carrier, delivery agent or excipient.
  • the disclosure provides a method of delivering an oligonucleotide to a cell or population of cells in the central nervous system, liver tissue, muscle tissue, adipose tissue, adrenal tissue and/or ocular tissue, the method comprising administering a pharmaceutical composition described herein.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of a target mRNA, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide or pharmaceutical composition described herein.
  • the disclosure provides a method of reducing expression of a target mRNA in a subject, comprising administering to the subject an oligonucleotide or pharmaceutical composition described herein.
  • the target mRNA is expressed in the liver. In some aspects, the target mRNA is expressed in a hepatocyte. In some aspects, the target mRNA is expressed in a liver sinusoidal endothelial cell. In some aspects of the methods described herein, the target mRNA is expressed in a macrophage of the liver. In some aspects, the target mRNA is expressed in at least one cell type of the central nervous system. In some aspects, the at least one cell type of the central nervous system is an astrocyte, a neuron, or an oligodendrocyte. In some aspects, the target mRNA is expressed in an astrocyte, a neuron, an oligodendrocyte, or any combination thereof.
  • the target mRNA is expressed in the frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, or any combination thereof. In some aspects of the methods described herein, the target mRNA is expressed in ocular tissue, optionally the retina or optic nerve. In some aspects of the methods described herein, the target mRNA is expressed in liver tissue, skeletal muscle tissue, adipose tissue and/or adrenal tissue.
  • the disclosure provides a kit comprising an oligonucleotide described herein, an optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder or condition associated with expression of a target mRNA.
  • the disclosure provides use of an oligonucleotide or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with expression of a target mRNA.
  • the disclosure provides an oligonucleotide or pharmaceutical composition described herein, for use, or adaptable for use, in the treatment of a disease, disorder, or condition associated with expression of a target mRNA.
  • the target mRNA is expressed in the liver, optionally wherein the target mRNA is expressed in a liver macrophage, a liver hepatocyte, or a liver sinusoidal endothelial cell. In any of the foregoing or related aspects, the target mRNA is expressed in ocular tissue, optionally the retina or optic nerve. In any of the foregoing or related aspects, the target mRNA is expressed in liver tissue, skeletal muscle tissue, adipose tissue and/or adrenal tissue. In some aspects, the target mRNA is expressed in at least one tissue of the central nervous system.
  • the at least one tissue of the central nervous system is selected from the frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combinations thereof.
  • the disease or disorder is selected from the group consisting of viral infections, bacterial infections, parasitic infections, cancers, allergies, autoimmune diseases, immunodeficiencies, and immunosuppression.
  • the disclosure provides a double-stranded oligonucleotide comprising:
  • a sense strand 14-20 nucleotides in length wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and
  • an antisense strand 22 nucleotides in length wherein the antisense strand comprises a region of complementarity to an astrocyte target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.
  • the disclosure provides a double-stranded oligonucleotide comprising: (i) a sense strand 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and
  • an antisense strand 22 nucleotides in length wherein the antisense strand comprises a region of complementarity to a neuron target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.
  • the disclosure provides a double-stranded oligonucleotide comprising:
  • a sense strand 14-20 nucleotides in length wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and
  • an antisense strand 22 nucleotides in length wherein the antisense strand comprises a region of complementarity to an oligodendrocyte target mRNA, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides.
  • the sense strand is 14 nucleotides in length.
  • the sense strand comprises a locked nucleic acid at one or more of position 2, position 9, position 10, position 12, or position 13 with positions numbered 5’ to 3’.
  • the antisense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 13 and 14, between positions 14 and 15, between positions 20 and 21, and between positions 21 and 22.
  • the sense strand is 20 nucleotides in length.
  • the sense strand comprises a locked nucleic acid at one or more of position 2, position 15, or position 16 with positions numbered 5’ to 3’.
  • the antisense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 2 and 3, between positions 3 and 4, between positions 20 and 21, and between positions 21 and 22.
  • the disclosure provides a method of reducing expression of a target mRNA in an astrocyte, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and
  • the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand.
  • the sense strand comprises no more than 3 locked nucleic acids. In some aspects, the sense strand is 20 nucleotides in length, and wherein reduction of the target mRNA in an astrocyte is increased compared to reduction in a neuron. In some aspects, reduction of the target mRNA is increased by at least 5%. In some aspects, reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%.
  • the sense strand is 14 nucleotides in length, and wherein reduction of the target mRNA in an astrocyte is increased compared to reduction in an oligodendrocyte. In some aspects, reduction of the target mRNA is increased by at least 5%. In some aspects, reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%. In some aspects, the sense strand is 20 nucleotides in length, and wherein the target mRNA is reduced in an astrocyte and an oligodendrocyte to the same or similar level.
  • the sense strand is 14 nucleotides in length, wherein the target mRNA is reduced in an astrocyte and in a neuron to the same or similar level, and wherein reduction of the target mRNA in an astrocyte and in a neuron is increased compared to reduction in an oligodendrocyte.
  • reduction of the target mRNA is increased by at least 5%.
  • reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%.
  • the disclosure provides a method of reducing expression of a target mRNA in an oligodendrocyte, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises: (i) a sense strand, wherein the sense strand is 14-40 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and
  • an antisense strand wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the oligodendrocyte, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the oligodendrocyte.
  • the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand.
  • the sense strand is 20 nucleotides in length.
  • the sense strand in 36 nucleotides in length, and wherein the oligonucleotide comprises a stem-loop.
  • the target mRNA is reduced in an astrocyte and the oligodendrocyte to the same or similar level, and wherein reduction of the target mRNA is increased in the astrocyte and the oligodendrocyte compared to reduction in a neuron. In some aspects, reduction of the target mRNA is increased by at least 5%.
  • reduction of the target mRNA is increased by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45% or by at least 50%.
  • the disclosure provides a method of reducing expression of a target mRNA in a neuron, comprising administering a double-stranded oligonucleotide wherein the oligonucleotide comprises:
  • a sense strand wherein the sense strand is 14-20 nucleotides in length, wherein the sense strand comprises at least one lipid moiety conjugated to the 5’ terminus, and wherein the sense strand comprises at least one locked nucleic acid, and
  • an antisense strand wherein the antisense strand is 22 nucleotides in length, and wherein the antisense strand comprises a region of complementarity to a target mRNA in the neuron, and wherein the sense and antisense strand are separate strands which form an asymmetric duplex region having an overhang of about 2-8 nucleotides at the 3’ terminus of the antisense strand, wherein the duplex region is about 14-20 nucleotides, thereby reducing expression of the target mRNA in the neuron.
  • the oligonucleotide comprises a blunt end comprising the 3’ end of the sense strand and the 5’ end of the antisense strand.
  • the sense strand comprises no more than 5 locked nucleic acids.
  • the sense strand is 14 nucleotides in length.
  • FIGs 1A-1C provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 1-14.
  • FIGs 2A-2F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 2A), hippocampus (FIG. 2B), cerebellum (FIG. 2C), brain stem (FIG. 2D), lumbar dorsal root ganglion (DRG) (FIG. 2E), and lumbar spinal cord (FIG. 2F) of control mice (group A) or mice administered Compounds 1-14 (respectively groups B-O) via lumbar intrathecal injection.
  • FIG. 3 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 15-18.
  • FIGs. 4A-4F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 4A), hippocampus (FIG. 4B), medulla (FIG. 4C) cerebellum (FIG. 4D), lumbar dorsal root ganglion (DRG) (FIG. 4E), and lumbar spinal cord (FIG. 4F) of control mice (group A) or mice administered Compounds 7-9 (respectively groups B-D) or Compounds 15-18 (respectively groups E-H) via lumbar intrathecal injection.
  • group A mice administered Compounds 7-9 (respectively groups B-D) or Compounds 15-18 (respectively groups E-H) via lumbar intrathecal injection.
  • FIG. 5 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 18-28.
  • FIGs. 6A-6B provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in cerebellum (FIG. 6A) and lumbar dorsal root ganglion (DRG) (FIG. 6B) of control mice (group A) or mice administered Compounds 18-28 (respectively groups B-L).
  • FIG. 7 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 18 and 29-38.
  • FIGs. 8A-8B provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in cerebellum (FIG. 8A) and lumbar dorsal root ganglion (DRG) (FIG. 8B) of control mice (group A) or mice administered Compounds 18 or 29-38 (respectively groups B- L).
  • FIG. 9 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting reticulon 4 (RTN4) mRNA having the structures of Compounds 39-49.
  • FIGs. 10A-10B provide graphs measuring percent (%) of rat RTN4 mRNA remaining in retina (FIG. 10A) and optic nerve (FIG. 8B) tissues of control rats (group A) or rats administered Compounds 39-49 (respectively groups B-L) via intravitreal injection.
  • FIG. 10C provides a schematic of RNAi oligonucleotide-GalNAc conjugates targeting Aldh2 mRNA having the structures of Compounds 57 and 98-108.
  • FIG. 10D provides a graph measuring percent (%) of murine Aldh2 mRNA remaining in liver tissue of control mice administered PBS (group A) or mice administered Compounds 57 or 98-108 (respectively groups B-N).
  • FIG. 10E provides a schematic of RNAi oligonucleotide-lipid conjugates targeting PEC AM- 1 mRNA having the structures of Compounds 120 and 121 and CD68 mRNA having the structure of Compound 122.
  • FIGs. 10F-10G provide graphs measuring percent (%) of murine PEC AM mRNA (FIG. 10F) and CD68 mRNA (FIG. 10G) remaining in liver tissue of control mice administered PBS (group A) or mice administered Compounds 120-122 (respectively groups B-D) via subcutaneous injection.
  • FIGs. 10H-10I provide graphs measuring percent (%) of murine CD68 mRNA (FIG. 10H) and CD68 protein (FIG. 101) remaining in liver tissue of control mice administered PBS or mice administered Compound 122 at the indicated dose via subcutaneous injection, as measured by qPCR and immunohistochemistry (IHC) respectively.
  • FIG. 10J provides a representative image of liver tissue obtained from a PBS control mice as described in FIGs. 10H-10I that were imaged following CD68 immunostaining. Arrows indicate representative cells that morphologically present as hepatocytes or macrophages. Dark staining indicates CD68 protein expression.
  • FIG. 10K provides representative images of liver tissue obtained from mice described in FIGs. 10H-10I that were imaged following CD68 immunostaining. Dark staining indicates CD68 protein expression.
  • FIGs. 11A-11B provide schematics of RNAi oligonucleotide-lipid conjugates targeting CD68 mRNA having the structures of Compounds 58-97.
  • FIG. 12 provides a graph measuring percent (%) of murine CD68 mRNA remaining in liver tissue of control mice administered PBS or mice administered Compounds 58-97 (respectively groups B-AO) via subcutaneous injection.
  • FIG. 13 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting Aldh2 mRNA having the structures of Compounds 109-117.
  • FIGs. 14A-14D provide graphs measuring percent of murine Aldh2 mRNA remaining in liver (FIG. 14A), adipose (FIG. 14B), skeletal muscle (FIG. 14C), and adrenal (FIG. 14D) tissues in control mice administered PBS (group A) or mice administered Compounds 109-117 (respectively groups B-J) via subcutaneous injection.
  • FIG. 15 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting STAT3 (Compounds 123 and 127), SLC25A1 (Compounds 124 and 128), HMGB1 (Compounds 125 and 129), and ALDH2 (Compounds 126 and 130).
  • FIGs. 16A-16D provide graphs measuring percent (%) of murine STAT3 mRNA remaining in liver tissue of mice administered PBS (group A) or compounds 123 or 127 (groups Bl and Cl respectively) (FIG. 16A); percent (%) of murine SLC25A1 mRNA remaining in liver tissue of mice administered PBS (group A) or compounds 124 or 128 (groups B2 and C2 respectively) (FIG. 16B); percent (%) of murine HMGB1 mRNA remaining in liver tissue of mice administered PBS (group A) or compounds 125 or 129 (groups B3 and C3 respectively) (FIG.
  • mice were administered RNAi oligonucleotidelipid conjugates via subcutaneous injection.
  • FIG. 17 provides a schematic of RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a P-4, P-6, or P-8 truncated sense strand (i.e. a 6, 8, and 10 nucleotide overhang of the antisense strand) (Compounds 131-133) or a p-4, p-6, or p-8 truncated sense strand comprising phosphorothioate linkages (Compounds 134-136).
  • a P-4, P-6, or P-8 truncated sense strand i.e. a 6, 8, and 10 nucleotide overhang of the antisense strand
  • Compounds 131-133 Compounds 131-133
  • a p-4, p-6, or p-8 truncated sense strand comprising phosphorothioate linkages
  • FIGs. 18A-18F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 18A), hippocampus (FIG. 18B), cerebellum (FIG. 18C), lumbar dorsal root ganglion (DRG) (FIG. 18D), medulla (FIG. 18E), and lumbar spinal cord (SC) (FIG. 18F) of control mice (aCSF) or mice administered Compounds 131-136 (respectively Parent Cl 6, P-4, P-6, P-8, P-4 PS, P-6 PS, and P-8 PS) via lumbar intrathecal injection.
  • FIG. 19 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a sense strand with different amounts of locked nucleic acids (LNA) (Compounds 137-140) or a P-6 truncated sense strand (i.e., an 8 nucleotide overhang of the antisense strand) comprising different amounts of LNAs (Compounds 141-145).
  • LNA locked nucleic acids
  • FIGs. 20A-20F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 20A), hippocampus (FIG. 20B), cerebellum (FIG. 20C), medulla (FIG. 20D), lumbar dorsal root ganglion (DRG) (FIG. 20E), and lumbar spinal cord (SC) (FIG. 20F) of control mice (aCSF) or mice administered Compounds 1 and 137-145 (respectively B-K) via lumbar intrathecal injection.
  • aCSF control mice
  • mice administered Compounds 1 and 137-145 (respectively B-K) via lumbar intrathecal injection.
  • FIG. 21 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 138, 139, 140, 147, and 148).
  • LNA locked nucleic acids
  • FIGs. 22A-22F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 22A), hippocampus (FIG. 22B), medulla (FIG. 22C), lumbar dorsal root ganglion (DRG) (FIG. 22D), cerebellum (FIG. 22E), and lumbar spinal cord (SC) (FIG. 22F) of control mice (aCSF) or mice administered Compounds 1, 137, 146, 138, 139, 140, 147, and 148 (respectively B-I) via lumbar intrathecal injection.
  • aCSF control mice
  • Compounds 1, 137, 146, 138, 139, 140, 147, and 148 respectively B-I
  • FIG. 23 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a P-4 truncated sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 149-154).
  • LNA locked nucleic acids
  • FIGs. 24A-24F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 24A), hippocampus (FIG. 24B), medulla (FIG. 24C), lumbar dorsal root ganglion (DRG) (FIG. 24D), cerebellum (FIG. 24E), and lumbar spinal cord (SC) (FIG. 24F) of control mice (aCSF) or mice administered Compounds 1, 137 and 149-154 (respectively B-I) via lumbar intrathecal injection.
  • aCSF control mice
  • mice administered Compounds 1, 137 and 149-154 (respectively B-I) via lumbar intrathecal injection.
  • FIG. 25 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises a P-8 truncated sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 155-160).
  • LNA locked nucleic acids
  • FIGs. 26A-26F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 26A), hippocampus (FIG. 26B), medulla (FIG. 26C), lumbar dorsal root ganglion (DRG) (FIG. 26D), cerebellum (FIG. 26E), and lumbar spinal cord (SC) (FIG. 26F) of control mice (aCSF) or mice administered Compounds 1, 137 and 155-160 (respectively B-I) via lumbar intrathecal injection.
  • aCSF control mice
  • mice administered Compounds 1, 137 and 155-160 respectively B-I
  • FIG. 27 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting UGT8 where the oligonucleotide comprises a sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 162-165) or a P-6 truncated sense strand comprising different amounts of LNAs (Compounds 166-171).
  • LNA locked nucleic acids
  • FIGs. 28A-28F provide graphs measuring percent (%) of murine UGT8 mRNA remaining in frontal cortex (FIG. 28A), hippocampus (FIG. 28B), hypothalamus (FIG. 28C), cerebellum (FIG. 28D), medulla (FIG. 28E), and lumbar spinal cord (FIG. 26F) of control mice (aCSF) or mice administered Compounds 161-171 (respectively B-L) via lumbar intrathecal injection.
  • FIG. 29 provides a schematic of blunt-end RNAi oligonucleotide-lipid conjugates targeting GFAP where the oligonucleotide comprises a sense strand comprising different amounts of locked nucleic acids (LNA) (Compounds 173-176) or a P-6 truncated sense strand comprising different amounts of LNAs (Compounds 177-182).
  • LNA locked nucleic acids
  • FIGs. 30A-30F provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 30A), hippocampus (FIG. 30B), cerebellum (FIG. 30C), medulla (FIG. 30D), hypothalamus (FIG. 30E), and lumbar spinal cord (FIG. 30F) of control mice (aCSF) or mice administered Compounds 172-182 (respectively B-L) via lumbar intrathecal injection.
  • FIGs. 31A-31D provide schematics of blunt-end RNAi oligonucleotide-lipid conjugates targeting TUBB3 where the oligonucleotide comprises different sense strand truncations including: no truncation (FIG. 31A; Compounds 137 and 183-190); a P-4 truncated sense strand (FIG. 31B; Compounds 149, and 191-194); a P-6 truncated sense strand (FIG. 31C; Compounds 141, 195, and 196); and, a P-8 truncated sense strand (FIG. 31D; Compound 197).
  • Each compound comprises a C16 lipid conjugated to a different position of the sense strand as indicated in the schematics.
  • FIGs. 32A-32F provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 32A), hippocampus (FIG. 32B), medulla (FIG. 32C), lumbar dorsal root ganglion (FIG. 32D), cerebellum (FIG. 32E), and lumbar spinal cord (FIG. 32F) of control mice (aCSF) or mice administered Compounds 137, 183-190, 149, 191-194, 141, and 195-197 via lumbar intrathecal injection.
  • aCSF control mice
  • mice administered Compounds 137, 183-190, 149, 191-194, 141, and 195-197 via lumbar intrathecal injection.
  • FIGs. 33A-33C provide schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 173, 176, 180, 200-205, and 207 -215.
  • FIGs. 34A-34D provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 34A), hippocampus (FIG. 34B), medulla (FIG. 34C), and lumbar spinal cord (FIG. 34D) of control mice (aCSF) or mice administered compounds 173, 176, 180, 200-205, and 207 -215 (as identified in Table 20) via lumbar intrathecal injection.
  • FIGs. 35A- 35E provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 137, 140, 144, 217-222, 224- 232, and 277-285.
  • FIGs. 36A-36D provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 34A), hippocampus (FIG. 34B), medulla (FIG. 34C), and lumbar spinal cord (FIG. 34D) of control mice (aCSF) or mice administered compounds 137, and 140, 144, 217-222, 224-232 (as identified in Table 21) via lumbar intrathecal injection.
  • FIG. 36E is a graph measuring (%) of murine TUBB3 mRNA remaining in frontal cortex hippocampus, hypothalamus, cerebellum, brain stem, and lumbar spinal cord of control mice (aCSF) or mice administered compounds 140, 277-278, 231, and 279-285 (respectively B-L) via lumbar intrathecal injection.
  • FIGs. 37A- 37B provide schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 173, 176, and 233-245.
  • FIGs. 38A-38D provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 38A), hippocampus (FIG. 38B), medulla (FIG. 38C), and lumbar spinal cord (FIG. 38D) of control mice (aCSF) or mice administered compounds 173, 176, and 233-245 (respectively B-P) via lumbar intrathecal injection.
  • FIGs. 39A- 39B provide schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 173, 176, 200 and 246-255.
  • FIGs. 40A-40D provide graphs measuring percent (%) of murine GFAP mRNA remaining in frontal cortex (FIG. 40A), hippocampus (FIG. 40B), medulla (FIG. 40C), and lumbar spinal cord (FIG. 40D) of control mice (aCSF) or mice administered compounds 173, 176, 200 and 246-255 (respectively B-N) via lumbar intrathecal injection.
  • FIGs. 41 A- 41B provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 137, 140, and 257-268.
  • FIGs. 42A-42D provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 42A), hippocampus (FIG. 42B), medulla (FIG. 42C), and lumbar spinal cord (FIG. 42D) of control mice (aCSF) or mice administered compounds 137, 140, and 257-268 (respectively B-P) via lumbar intrathecal injection.
  • FIGs. 43 A- 43B provide schematics of RNAi oligonucleotide-lipid conjugates targeting TUBB3 mRNA having the structures of Compounds 137, 140, 217, and 269-276.
  • FIGs. 44A-44D provide graphs measuring percent (%) of murine TUBB3 mRNA remaining in frontal cortex (FIG. 44A), hippocampus (FIG. 44B), medulla (FIG. 44C), and lumbar spinal cord (FIG. 44D) of control mice (aCSF) or mice administered compounds 137, 140, 217, and 269-276 (respectively B-F and I-N) via lumbar intrathecal injection.
  • FIG. 45 provides schematics of RNAi oligonucleotide-lipid conjugates targeting GFAP mRNA having the structures of Compounds 286-292.
  • FIGs. 46A-46D provide graphs measuring percent (%) of murine ALDH2 mRNA remaining in liver (FIG. 46A), quadricep (quad) muscle (FIG. 46B), heart muscle (FIG. 46C), and gonadal white adipose tissue (gWAT) (FIG. 46D) of control mice (PBS) or mice administered compounds 286-292 (respectively A-G) via subcutaneous injection.
  • FIG. 47 provides schematics of RNAi oligonucleotide-lipid conjugates targeting UGT8 mRNA having the structures of Compounds 162, 167, 293, and 294.
  • FIGs. 48A-48D provide graphs measuring percent (%) of murine UGT8 mRNA remaining in frontal cortex (FIG. 48A), hippocampus (FIG. 48B), brain stem (FIG. 48C), and lumbar spinal cord (FIG. 48D) of control mice (aCSF) or mice administered compounds 162, 167, 293, and 294 (respectively blunt, 5’ p-6, 3’ p-3, and 5’ p-6 & 3’ p-3) via lumbar intrathecal injection.
  • the disclosure provides oligonucleotide-lipid conjugates (e.g., RNAi oligonucleotide-lipid conjugates) that reduce expression of a target gene.
  • the disclosure provides methods of treating a disease or disorder associated with expression of a target gene.
  • the disclosure provides methods of treating a disease or disorder (e.g., a neurological disease and/or by inappropriate gene expression) associated with expression of a target gene using the lipid-conjugated RNAi oligonucleotides, or pharmaceutically acceptable compositions thereof, described herein.
  • the disclosure provides methods of using the lipid-conjugated RNAi oligonucleotides described herein in the manufacture of a medicament for treating a disease or disorder associated with expression of a target gene.
  • BNAs bridged nucleic acids
  • LNA locked nucleic acids
  • thermostability provided by LNA’s and BNA’s allow modifications of underlying RNAi structures. For example, significant truncations in the passenger/sense strand of a given trigger can be made and with the appropriate addition and use of LNA’s or BNA’s the gene knockdown activity of every shorter passenger strands can be rescued. The result is a LNS enhanced RNAi trigger that is lighter in weight with the equivalent activity in physiological systems.
  • phosphorothioate molecules can be used to control or lessen nuclease attack on a given RNAi trigger structure. In this sense the chemical modification of a given RNAi trigger can be a balancing act between those modifications such as LNA, BNA or Phosphorothioate (PS) placement to protect against nuclease degradation or loss of thermostability.
  • RNAi oligonucleotides e.g., RNAi oligonucleotide-lipid conjugates
  • a lipid-conjugated RNAi oligonucleotide provided by the disclosure is targeted to an mRNA encoding the target gene.
  • Messenger RNA (mRNA) that encodes a target gene and is targeted by a lipid-conjugated RNAi oligonucleotide of the disclosure is referred to herein as “target mRNA”.
  • the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence within a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide, or a portion, fragment, or strand thereof (e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) binds or anneals to a target sequence comprising a target mRNA, thereby reducing target gene expression.
  • a target sequence comprising a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence within a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotide, or
  • the lipid-conjugated RNAi oligonucleotide is targeted to a target sequence comprising target mRNA for the purpose of reducing expression of a target gene in vivo.
  • the amount or extent of reduction of target gene expression by a lipid- conjugated RNAi oligonucleotide targeted to a specific target sequence correlates with the potency of the lipid-conjugated RNAi oligonucleotide.
  • the amount or extent of reduction of target gene expression by a lipid-conjugated RNAi oligonucleotide targeted to a specific target sequence correlates with the amount or extent of therapeutic benefit in a subject or patient having a disease, disorder or condition associated with target gene expression treated with the lipid-conjugated RNAi oligonucleotide.
  • nucleotide sequence of mRNAs encoding target genes including mRNAs of multiple different species (e.g., human, cynomolgus monkey, mouse, and rat) and as a result of in vitro and in vivo testing, it has been discovered that certain nucleotide sequences and certain systemic modifications to those oligonucleotides are more amenable than others to RNAi oligonucleotide-mediated reduction and are thus useful as part of oligonucleotides that are otherwise targeted to specific gene target sequences.
  • mRNAs of multiple different species e.g., human, cynomolgus monkey, mouse, and rat
  • a sense strand of a lipid-conjugated RNAi oligonucleotide, or a portion or fragment thereof, described herein comprises a nucleotide sequence that is similar (e.g. , having no more than 4 mismatches) or is identical to a target sequence comprising a target mRNA.
  • a portion or region of the sense strand of a double-stranded oligonucleotide described herein comprises a target sequence comprising a target mRNA.
  • the target mRNA is expressed in a tissue or cell of a subject. In some embodiments, the target mRNA is expressed in more than one tissue or cell of a subject, wherein the tissues or cells are different. In some embodiments, the target mRNA is differentially expressed in a tissue or cell. In some embodiments, the target mRNA is expressed throughout multiple tissue types and/or cell types. In some embodiments, the target mRNA is expressed in the central nervous system, liver tissue, ocular tissue, adipose tissue, adrenal tissue, or skeletal muscle tissue.
  • the target mRNA is expressed in the central nervous system, peripherial nervous system, liver tissue, ocular tissue, adipose tissue, adrenal tissue, heart tissue, lung tissue, skeletal muscle tissue, cardiac muscle tissue, smooth muscle tissue, or kidney tissue, or any combination thereof.
  • the target mRNA is expressed in the central nervous system.
  • the target mRNA is expressed in the peripherial nervous system.
  • the target mRNA is expressed in a region of the central nervous system.
  • a region of the central nervous system is selected from the eye, brain, cerebrum, cerebral cortex, frontal lobe, frontal cortex, parietal lobe, temporal lobe, occipital lobe, hippocampus, cerebellum, brain stem, dorsal root ganglion (DRG) or the spinal cord.
  • the target mRNA is expressed in a neuron.
  • the target mRNA is expressed in a glial cell.
  • the target mRNA is expressed in a neuron located in the central nervous system.
  • the target mRNA is expressed in liver tissue.
  • the target mRNA is expressed in a hepatocyte.
  • the target mRNA is expressed in a liver sinusoidal endothelial cell. In some embodiments, the target mRNA is expressed in a macrophage. In some embodiments, the target mRNA is expressed in a macrophage located in liver tissue. In some embodiments, the target mRNA is expressed in ocular tissue. In some embodiments, the target mRNA is expressed in the retina and/or optic nerve. In some embodiments, the target mRNA is expressed in adipose tissue. In some embodiments, the target mRNA is expressed in skeletal muscle tissue. In some embodiments, the target mRNA is expressed in adrenal tissue. In some embodiments, the target mRNA is expressed in heart tissue.
  • the target mRNA is expressed in lung tissue. In some embodiments, the target mRNA is expressed in the eye. In some embodiments, the target mRNA is expressed in the brain. In some embodiments, the target mRNA is expressed in the cerebrum. In some embodiments, the target mRNA is expressed in the cerebellum. In some embodiments, the target mRNA is expressed in the brain stem. In some embodiments, the target mRNA is expressed in the frontal lobe. In some embodiments, the target mRNA is expressed in the frontal cortex. In some embodiments, the target mRNA is expressed in the parietal lobe. In some embodiments, the target mRNA is expressed in the temporal lobe.
  • the target mRNA is expressed in the occipital lobe. In some embodiments, the target mRNA is expressed in the hippocampus. In some embodiments, the target mRNA is expressed in the DRG. In some embodiments, the target mRNA is expressed in the spinal cord.
  • the lipid-conjugated RNAi oligonucleotides provided by the disclosure comprise a targeting sequence.
  • targeting sequence refers to a nucleotide sequence having a region of complementarity to a specific nucleotide sequence comprising an mRNA.
  • the lipid-conjugated RNAi oligonucleotides provided by the disclosure comprise a gene targeting sequence having a region of complementarity to a nucleotide sequence comprising a target sequence of a target mRNA.
  • the targeting sequence imparts the lipid-conjugated RNAi oligonucleotide with the ability to specifically target an mRNA by binding or annealing to a target sequence comprising a target mRNA by complementary (Watson-Crick) base pairing.
  • the lipid-conjugated RNAi oligonucleotides herein (or a strand thereof, e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) comprise a targeting sequence having a region of complementarity that binds or anneals to a target sequence comprising a target mRNA by complementary (Watson-Crick) base pairing.
  • the lipid-conjugated RNAi oligonucleotides herein (or a strand thereof, e.g., an antisense strand or a guide strand of a double-stranded oligonucleotide) comprise a targeting sequence having a region of complementarity that binds or anneals to a target sequence within a target mRNA by complementary (Watson-Crick) base pairing.
  • the targeting sequence is generally of suitable length and base content to enable binding or annealing of the lipid-conjugated RNAi oligonucleotide (or a strand thereof) to a specific target mRNA for purposes of inhibiting target gene expression.
  • the targeting sequence is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29 or at least about 30 nucleotides in length.
  • the targeting sequence is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides.
  • the targeting sequence is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides in length.
  • the targeting sequence is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the targeting sequence is 18 nucleotides in length. In some embodiments, the targeting sequence is 19 nucleotides in length. In some embodiments, the targeting sequence is 20 nucleotides in length. In some embodiments, the targeting sequence is 21 nucleotides in length. In some embodiments, the targeting sequence is 22 nucleotides in length. In some embodiments, the targeting sequence is 23 nucleotides in length. In some embodiments, the targeting sequence is 24 nucleotides in length.
  • the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence comprising a target mRNA. In some embodiments, the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence within a target mRNA. In some embodiments, the targeting sequence is partially complementary to a target sequence comprising a target mRNA. In some embodiments, the targeting sequence is partially complementary to a target sequence within a target mRNA. In some embodiments, the targeting sequence comprises a region of contiguous nucleotides comprising the antisense strand.
  • the lipid-conjugated RNAi oligonucleotides herein comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20 or 18 to 19 nucleotides in length).
  • the lipid- conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.
  • the lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 15 nucleotides in length.
  • the lipid-conjugated RNAi oligonucleotides comprise a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length.
  • the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 15 nucleotides in length. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence that is complementary to a contiguous sequence of nucleotides comprising a target mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising a target mRNA and comprises the entire length of an antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising a target mRNA and comprises a portion of the entire length of an antisense strand.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising a target mRNA and comprises 10 to 20 nucleotides of the antisense strand.
  • a targeting sequence of a lipid- conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising a target mRNA and comprises 15 to 19 nucleotides of the antisense strand.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising a target mRNA and comprises 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides of the antisense strand.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is fully complementary (e.g., having no mismatches) to a target sequence comprising a target mRNA and comprises 19 nucleotides of the antisense strand.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g. , having no more than 4 mismatches) to a target sequence comprising a target mRNA and comprises the entire length of an antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising a target mRNA and comprises a portion of the entire length of an antisense strand.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising a target mRNA and comprises 10 to 20 nucleotides of the antisense strand. In some embodiments, a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising a target mRNA and comprises 15 to 19 nucleotides of the antisense strand.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising a target mRNA and comprises 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides of the antisense strand.
  • a targeting sequence of a lipid-conjugated RNAi oligonucleotide herein is partially complementary (e.g., having no more than 4 mismatches) to a target sequence comprising a target mRNA and comprises 19 nucleotides of the antisense strand.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a targeting sequence having one or more base pair (bp) mismatches with the corresponding target sequence comprising a target mRNA.
  • the targeting sequence has a 1 bp mismatch, a 2 bp mismatch, a 3 bp mismatch, a 4 bp mismatch, or a 5 bp mismatch with the corresponding target sequence comprising a target mRNA provided that the ability of the targeting sequence to bind or anneal to the target sequence under appropriate hybridization conditions and/or the ability of the lipid-conjugated RNAi oligonucleotide to inhibit or reduce target gene expression is maintained (e.g., under physiological conditions).
  • the targeting sequence comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 bp mismatches with the corresponding target sequence comprising a target mRNA provided that the ability of the targeting sequence to bind or anneal to the target sequence under appropriate hybridization conditions and/or the ability of the lipid-conjugated RNAi oligonucleotide to inhibit or reduce target gene expression is maintained.
  • the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 1 mismatch with the corresponding target sequence.
  • the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 2 mismatches with the corresponding target sequence. In some embodiments, the lipid- conjugated RNAi oligonucleotide comprises a targeting sequence having 3 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 4 mismatches with the corresponding target sequence. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having 5 mismatches with the corresponding target sequence.
  • the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or wherein the mismatches are interspersed in any position throughout the targeting sequence.
  • mismatch e.g., 2, 3, 4, 5 or more mismatches
  • the lipid-conjugated RNAi oligonucleotide comprises a targeting sequence having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least 2 (e.g., all) of the mismatches are positioned consecutively (e.g., 2, 3, 4, 5 or more mismatches in a row), or wherein at least one or more non-mismatched base pair is located between the mismatches, or a combination thereof.
  • mismatch e.g., 2, 3, 4, 5 or more mismatches
  • RNAi oligonucleotide types and/or structures are useful for reducing target gene expression in the methods herein. Any of the RNAi oligonucleotide types described herein or elsewhere are contemplated for use as a framework to incorporate a targeting sequence herein for the purposes of inhibiting or reducing corresponding target gene expression.
  • the lipid-conjugated RNAi oligonucleotides herein inhibit target gene expression by engaging with RNA interference (RNAi) pathways upstream or downstream of Dicer involvement.
  • RNAi RNA interference
  • RNAi oligonucleotides have been developed with each strand having sizes of about 19-25 nucleotides with at least one 3' overhang of 1 to 5 nucleotides (see, e.g., US Patent No. 8,372,968). Longer oligonucleotides also have been developed that are processed by Dicer to generate active RNAi products (see, e.g., US Patent No. 8,883,996).
  • extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically stabilizing tetraloop structure (see, e.g., US Patent Nos. 8,513,207 and 8,927,705, as well as Inti. Patent Application Publication No. WO 2010/033225).
  • Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.
  • the RNAi oligonucleotides conjugates herein engage with the RNAi pathway downstream of the involvement of Dicer (e.g., Dicer cleavage).
  • the oligonucleotides described herein are Dicer substrates.
  • the oligonucleotides herein interact with Dicer and are loaded into RISC.
  • double-stranded nucleic acids of 19- 23 nucleotides in length capable of reducing expression of a target mRNA are produced upon endogenous Dicer processing.
  • the lipid-conjugated RNAi oligonucleotide has an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3' end of the sense strand.
  • the lipid- conjugated RNAi oligonucleotide e.g., siRNA conjugate
  • oligonucleotide designs also are contemplated including oligonucleotides having a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, where there is a blunt end on the right side of the molecule (3' end of passenger strand/5' end of guide strand) and a two nucleotide 3 '-guide strand overhang on the left side of the molecule (5 ' end of the passenger strand/3' end of the guide strand). In such molecules, there is a 21 bp duplex region. See, e.g., US Patent Nos. 9,012,138; 9,012,621; 9,193,753; 8,420,391; and, 8,552,171 all to Tuschl et al. Such patents also indicate a lack of activity with regard to double overhang constructs.
  • the RNAi oligonucleotides conjugates disclosed herein comprise sense and antisense strands that are both in the range of about 17 to 26 (e.g., 17 to 26, 20 to 25 or 21-23) nucleotides in length.
  • the lipid-conjugated RNAi oligonucleotides disclosed herein comprise a sense and antisense strand that are both in the range of about 19-22 nucleotides in length.
  • the sense and antisense strands are of equal length.
  • the lipid-conjugated RNAi oligonucleotides disclosed herein comprise sense and antisense strands, such that there is a 3 '-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand.
  • a 3' overhang on the sense, antisense, or both sense and antisense strands is 1 or 2 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide has a guide strand of 22 nucleotides and a passenger strand of 20 nucleotides, where there is a blunt end on the right side of the molecule (3' end of passenger strand/5' end of guide strand) and a 2 nucleotide 3 '-guide strand overhang on the left side of the molecule (5' end of the passenger strand/3' end of the guide strand). In such molecules, there is a 20 bp duplex region.
  • RNAi oligonucleotide designs for use with the compositions and methods herein include: 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology, Blackburn (ed.), ROYAL SOCIETY OF CHEMISTRY, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. (2010) METHODS MOL. BIOL. 629: 141-58), blunt siRNAs (e.g., of 19 bps in length; see, e.g., Kraynack & Baker (2006) RNA 12: 163-76), asymmetrical siRNAs (aiRNA; see, e.g., Sun etal.
  • siRNAs see, e.g., Nucleic Acids in Chemistry and Biology, Blackburn (ed.), ROYAL SOCIETY OF CHEMISTRY, 2006
  • shRNAs e.g., having 19 bp or shorter stems
  • siRNA small internally segmented interfering RNA
  • oligonucleotide structure that may be used in some embodiments to reduce or inhibit the expression of a target gene are microRNA (miRNA), short hairpin RNA (shRNA) and short siRNA (see, e.g., Hamilton etal. (2002) EMBO J. 21 :4671-79; see also, US Patent Application Publication No. 2009/0099115).
  • miRNA microRNA
  • shRNA short hairpin RNA
  • siRNA see, e.g., Hamilton etal. (2002) EMBO J. 21 :4671-79; see also, US Patent Application Publication No. 2009/0099115.
  • an antisense strand of a lipid-conjugated RNAi oligonucleotide is referred to as a “guide strand.”
  • a guide strand an antisense strand that engages with RNA-induced silencing complex (RISC) and binds to an Argonaute protein such as Ago2, or engages with or binds to one or more similar factors, and directs silencing of a target gene
  • RISC RNA-induced silencing complex
  • Ago2 Argonaute protein
  • a sense strand complementary to a guide strand is referred to as a “passenger strand.”
  • a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, up to 15, or up to 8 nucleotides in length).
  • a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35 or at least 38 nucleotides in length).
  • a herein comprises an antisense strand in a range of about 8 to about 40 (e.g., 8 to 40, 8 to 36, 8 to 32, 8 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 30, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of 15 to 30 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises an antisense strand of 12 to 30 nucleotides in length.
  • an antisense strand of any one of the lipid-conjugated RNAi oligonucleotide disclosed herein is of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.
  • a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 19-23 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 19 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 20 nucleotides in length. In some embodiments, a lipid- conjugated RNAi oligonucleotide comprises an antisense strand of 21 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 22 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide comprises an antisense strand of 23 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand (or passenger strand) of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17 or up to 12 nucleotides in length).
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36 or at least 38 nucleotides in length).
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of at least about 10 nucleotides in length (e.g., at least 10, at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36 or at least 38 nucleotides in length).
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand in a range of about 12 to about 50 (e.g., 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides in length.
  • 12 to about 50 e.g., 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand in a range of about 10 to about 50 (e.g., 10 to 50, 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40 or 32 to 40) nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand 15 to 50 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand 18 to 38 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 12-21 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 10 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 11 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 12 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 13 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 14 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 15 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 16 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 17 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 18 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 19 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 20 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 21 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 22 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 23 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 24 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 25 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 26 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 27 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 28 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 29 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 30 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 31 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 32 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 33 nucleotides in length.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 34 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 35 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 36 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 37 nucleotides in length. In some embodiments, a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand of 38 nucleotides in length.
  • a sense strand comprises a stem-loop structure at its 3' end. In some embodiments, a sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem-loop is formed by intrastrand base pairing. In some embodiments, a sense strand comprises a stem-loop structure at its 5' end. In some embodiments, a stem is a duplex of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 1 nucleotide in length. In some embodiments, the stem of the stem-loop comprises a duplex of 2 nucleotides in length.
  • the stem of the stem-loop comprises a duplex of 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 8 nucleotides in length.
  • the stem of the stem-loop comprises a duplex of 9 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 10 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 12 nucleotides in length. In some embodiments, the stem of the stem -loop comprises a duplex of 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 14 nucleotides in length.
  • a stem-loop provides the lipid-conjugated RNAi oligonucleotide protection against degradation (e.g., enzymatic degradation), facilitates or improves targeting and/or delivery to a target cell, tissue, or organ, or both.
  • the loop of a stem-loop provides nucleotides comprising one or more modifications that facilitate, improve, or increase targeting to a target mRNA (e.g., a target mRNA expressed in the CNS), inhibition of target gene expression, and/or delivery to a target cell, tissue, or organ (e.g., the CNS), or a combination thereof.
  • the stemloop itself or modification(s) to the stem-loop do not substantially affect the inherent gene expression inhibition activity of the lipid-conjugated RNAi oligonucleotide, but facilitates, improves, or increases stability (e.g., provides protection against degradation) and/or delivery of the lipid-conjugated RNAi oligonucleotide to a target cell, tissue, or organ (e.g., the CNS).
  • a lipid-conjugated RNAi oligonucleotide herein comprises a sense strand comprising (e.g., at its 3' end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a single-stranded loop between SI and S2 of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length).
  • the loop (L) is 3 nucleotides in length. In some embodiments, the loop (L) is 4 nucleotides in length.
  • the tetraloop comprises the sequence 5’-GAAA-3’. In some embodiments, the tetraloop comprises the sequence 5’-UNCG-3’. In some embodiments, the tetraloop comprises the sequence 5’-UACG-3’. In some embodiments, the stem loop comprises the sequence 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 526).
  • a loop (L) of a stem -loop having the structure S1-L-S2 as described above is a triloop.
  • the triloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.
  • a loop (L) of a stem -loop having the structure S1-L-S2 as described above is a tetraloop (e.g., within a nicked tetraloop structure).
  • the tetraloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof.
  • a loop (L) of a stem -loop having the structure S1-L-S2 as described above is a tetraloop as described in US Patent No. 10,131,912, incorporated herein by reference (e.g., within a nicked tetraloop structure).
  • Duplex Length is a tetraloop as described in US Patent No. 10,131,912, incorporated herein by reference (e.g., within a nicked tetraloop structure).
  • a duplex formed between a sense and antisense strand is at least 8 (e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16) nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is at least 12 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length.
  • a duplex formed between a sense and antisense strand is in the range of 12-30 nucleotides in length (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and antisense strand is in the range of 10-30 nucleotides in length (e.g., 10 to 30, 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides in length).
  • a duplex formed between a sense and antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, a duplex formed between a sense and antisense strand is 10-18 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 15-30 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 17-21 base pairs in length.
  • a duplex formed between a sense and antisense strand is 10 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 11 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 12 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 13 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 14 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 15 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 16 base pairs in length.
  • a duplex formed between a sense and antisense strand is 17 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 18 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 19 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 20 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand is 21 base pairs in length. In some embodiments, a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and/or antisense strand.
  • a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In some embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.
  • a lipid-conjugated RNAi oligonucleotide disclosed herein comprises sense and antisense strands, such that there is a 3 ’-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand.
  • a lipid-conjugated RNAi oligonucleotide herein has one 5’end that is thermodynamically less stable compared to the other 5’ end.
  • an asymmetric lipid-conjugated RNAi oligonucleotide conjugate is provided that includes a blunt end at the 3 ’end of a sense strand and overhang at the 3’ end of the antisense strand.
  • a 3’ overhang on an antisense strand is 1-4 nucleotides in length (e.g., 1, 2, 3, or 4 nucleotides in length).
  • the 3’-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length). In some embodiments, the 3’ overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one
  • the 3’-overhang is about two (2) to about twelve (12) nucleotides in length. In some embodiments, the 3’-overhang is (1) nucleotide in length. In some embodiments, the 3 ’-overhang is two (2) nucleotides in length. In some embodiments, the 3 ’-overhang is three (3) nucleotides in length. In some embodiments, the 3 ’-overhang is four (4) nucleotides in length. In some embodiments, the 3’- overhang is five (5) nucleotides in length. In some embodiments, the 3 ’-overhang is six (6) nucleotides in length.
  • the 3 ’-overhang is seven (7) nucleotides in length. In some embodiments, the 3 ’-overhang is eight (8) nucleotides in length. In some embodiments, the 3 ’-overhang is nine (9) nucleotides in length. In some embodiments, the 3’- overhang is ten (10) nucleotides in length. In some embodiments, the 3’-overhang is eleven
  • the 3’-overhang is twelve (12) nucleotides in length. In some embodiments, the 3’-overhang is thirteen (13) nucleotides in length. In some embodiments, the 3’-overhang is fourteen (14) nucleotides in length. In some embodiments, the 3 ’-overhang is fifteen (15) nucleotides in length. In some embodiments, the 3 ’-overhang is sixteen (16) nucleotides in length. In some embodiments, the 3 ’-overhang is seventeen (17) nucleotides in length. In some embodiments, the 3 ’-overhang is eighteen (18) nucleotides in length. In some embodiments, the 3’-overhang is nineteen (19) nucleotides in length. In some embodiments, the 3 ’-overhang is twenty (20) nucleotides in length.
  • an oligonucleotide for RNAi has a two (2) nucleotide overhang on the 3 ’ end of the antisense (guide) strand.
  • an overhang is a 3’ overhang comprising a length of between one and four nucleotides, optionally one to four, one to three, one to two, two to four, two to three, or one, two, three, or four nucleotides.
  • the overhang is a 5’ overhang comprising a length of between one and four nucleotides, optionally one to four, one to three, one to two, two to four, two to three, or one, two, three, or four nucleotides.
  • an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the 5’ terminus of either or both strands comprise a 5 ’-overhang comprising one or more nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the sense strand comprises a 5’- overhang comprising one or more nucleotides. In some embodiments, an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 5 ’-overhang comprising one or more nucleotides.
  • an oligonucleotide herein comprises a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprises a 5 ’-overhang comprising one or more nucleotides.
  • the 5’-overhang is about one (1) to twenty (20) nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 nucleotides in length).
  • the 5’ overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides in length.
  • the 5’-overhang is (1) nucleotide in length. In some embodiments, the 5 ’-overhang is two (2) nucleotides in length. In some embodiments, the 5 ’-overhang is three (3) nucleotides in length. In some embodiments, the 5 ’-overhang is four (4) nucleotides in length. In some embodiments, the 5 ’-overhang is five (5) nucleotides in length. In some embodiments, the 5 ’-overhang is six (6) nucleotides in length. In some embodiments, the 5 ’-overhang is seven (7) nucleotides in length. In some embodiments, the 5 ’-overhang is eight (8) nucleotides in length.
  • the 5’-overhang is nine (9) nucleotides in length. In some embodiments, the 5’-overhang is ten (10) nucleotides in length. In some embodiments, the 5’-overhang is eleven (11) nucleotides in length. In some embodiments, the 5’-overhang is twelve (12) nucleotides in length. In some embodiments, the 5 ’-overhang is thirteen (13) nucleotides in length. In some embodiments, the 5’-overhang is fourteen (14) nucleotides in length. In some embodiments, the 5’-overhang is fifteen (15) nucleotides in length. In some embodiments, the 5’-overhang is sixteen (16) nucleotides in length.
  • the 5’-overhang is seventeen (17) nucleotides in length. In some embodiments, the 5’-overhang is eighteen (18) nucleotides in length. In some embodiments, the 5 ’-overhang is nineteen (19) nucleotides in length. In some embodiments, the 5 ’-overhang is twenty (20) nucleotides in length.
  • the 5’ overhang is 2 nucleotides and the 3’ overhang is about 3-7 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 3 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 4 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 5 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 6 nucleotides. In some embodiments, the 5’ overhang is 2 nucleotides and the 3’ overhang is 7 nucleotides.
  • the 3’ overhang is 6-8 nucleotides and the 5’ overhang is 2-4 nucleotides. In some embodiments, the 3’ overhang is 6 nucleotides and the 5’ overhang is 2 nucleotides. In some embodiments, the 3’ overhang is 6 nucleotides and the 5’ overhang is 3 nucleotides. In some embodiments, the 3’ overhang is 6 nucleotides and the 5’ overhang is 4 nucleotides. In some embodiments, the 3’ overhang is 7 nucleotides and the 5’ overhang is 2 nucleotides. In some embodiments, the 3’ overhang is 7 nucleotides and the 5’ overhang is 3 nucleotides.
  • the 3’ overhang is 7 nucleotides and the 5’ overhang is 4 nucleotides. In some embodiments, the 3’ overhang is 8 nucleotides and the 5’ overhang is 2 nucleotides. In some embodiments, the 3’ overhang is 8 nucleotides and the 5’ overhang is 3 nucleotides. In some embodiments, the 3’ overhang is 8 nucleotides and the 5’ overhang is 4 nucleotides. In some embodiments, one or more (e.g., 2, 3, or 4) terminal nucleotides of the 3’ end or 5’ end of a sense and/or antisense strand are modified.
  • one or two terminal nucleotides of the 3’ end of the antisense strand are modified.
  • the last nucleotide at the 3’ end of an antisense strand is modified, e.g., comprises 2’ modification, e.g., a 2’-O-methoxyethyl.
  • the last one or two terminal nucleotides at the 3’ end of an antisense strand are complementary with the target.
  • the last one or two nucleotides at the 3’ end of the antisense strand are not complementary with the target.
  • an RNAi oligonucleotide conjugate disclosed herein comprises a stem-loop structure at the 3’ end of the sense strand and comprises two terminal overhang nucleotides at the 3’ end of the antisense strand.
  • an RNAi oligonucleotide conjugate herein comprises a nicked tetraloop structure, wherein the 3’ end of the sense strand comprises a stem-tetraloop structure and comprises two terminal overhang nucleotides at the 3’ end of the antisense strand.
  • an RNAi oligonucleotide conjugate disclosed herein comprises a stem-loop structure at the 5’ end of the sense strand and comprises an overhang nucleotides at the 3’ end of the antisense strand.
  • an RNAi oligonucleotide conjugate herein comprises a nicked tetraloop structure, wherein the 5’ end of the sense strand comprises a stem-tetraloop structure and comprises two terminal overhang nucleotides at the 3’ end of the antisense strand.
  • an RNAi oligonucleotide conjugate disclosed herein comprises a stem-loop structure at the 5’ end of the sense strand and comprises a blunt end at the 5’ end of the antisense strand.
  • an RNAi oligonucleotide conjugate disclosed herein comprises an overhang of 1-8 nucleotides at the 5’ end of the sense strand and comprises an overhang of 1-8 nucleotides at the 5’ end of the antisense strand.
  • the overhang is selected from AA, GG, AG, and GA. In some embodiments, the overhang is AA. In some embodiments, the overhang is AG. In some embodiments, the overhang is GA. In some embodiments, the two terminal overhang nucleotides are GG. Typically, one or both of the two terminal GG nucleotides of the antisense strand are not complementary with the target.
  • the 5’ end and/or the 3 ’end of a sense or antisense strand has an inverted cap nucleotide.
  • one or more (e.g., 2, 3, 4, 5, 6) modified intemucleotide linkages are provided between terminal nucleotides of the 3’ end or 5’ end of a sense and/or antisense strand.
  • modified intemucleotide linkages are provided between overhang nucleotides at the 3’ end or 5’ end of a sense and/or antisense strand.
  • the sense strand is 18 nucleotides, and the antisense strand comprises a 5 ’overhang of 2 nucleotides, and a 3 ’overhang of 2 nucleotides. In some embodiments, the sense strand is 17 nucleotides, and the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 2 nucleotides. In some embodiments, the sense strand is 16 nucleotides, and the antisense strand comprises a 5’overhang of 4 nucleotides, and a 3’overhang of 2 nucleotides.
  • the sense strand is 13 nucleotides, and the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 7 nucleotides.
  • the sense strand is 12 nucleotides, and the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 8 nucleotides.
  • the sense strand is 12 nucleotides, and the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 7 nucleotides.
  • the sense strand is 10 nucleotides, and the antisense strand comprises a 5’overhang of 1 nucleotide, and a 3’overhang of 11 nucleotides.
  • the sense strand is 18 nucleotides
  • the duplex region is 18 nucleotides
  • the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 2 nucleotides.
  • the sense strand is 17 nucleotides
  • the duplex region is 17 nucleotides
  • the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 2 nucleotides.
  • the sense strand is 16 nucleotides
  • the duplex region is 16 nucleotides
  • the antisense strand comprises a 5’overhang of 4 nucleotides, and a 3’overhang of 2 nucleotides.
  • the sense strand is 13 nucleotides
  • the duplex region is 13 nucleotides
  • the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 7 nucleotides.
  • the sense strand is 12 nucleotides
  • the duplex region is 12 nucleotides
  • the antisense strand comprises a 5’overhang of 2 nucleotides, and a 3’overhang of 8 nucleotides.
  • the sense strand is 12 nucleotides
  • the duplex region is 12 nucleotides
  • the antisense strand comprises a 5’overhang of 3 nucleotides, and a 3’overhang of 7 nucleotides.
  • the sense strand is 10 nucleotides
  • the duplex region is 10 nucleotides
  • the antisense strand comprises a 5’overhang of 1 nucleotide, and a 3’overhang of 11 nucleotides.
  • an RNAi oligonucleotide conjugate disclosed herein comprises one or more modifications.
  • Oligonucleotides e.g., RNAi oligonucleotides
  • the modification is a modified sugar.
  • the modification is a 5’-terminal phosphate group.
  • the modification is a modified internucleoside linkage.
  • the modification is a modified base.
  • an oligonucleotide described herein can comprise any one of the modifications described herein or any combination thereof.
  • an oligonucleotide described herein comprises at least one modified sugar, a 5’- terminal phosphate group, at least one modified internucleoside linkage, and at least one modified base.
  • oligonucleotide e.g., an RNAi oligonucleotide
  • oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier.
  • LNP lipid nanoparticle
  • an oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Accordingly, in some embodiments, all or substantially all of the nucleotides of an oligonucleotides are modified.
  • the sugar moiety of all nucleotides comprising the oligonucleotide is modified at the 2’ position. In some embodiments, the sugar moiety of all nucleotides comprising the oligonucleotide is modified at the 2’ position, except for the nucleotide conjugated to a lipid (e.g., the 5 ’-terminal nucleotide of the sense strand). The modifications may be reversible or irreversible.
  • an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristics (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).
  • a nucleotide modification in a sugar comprises a 2'- modification.
  • a 2'-modification may be 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'- O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA) or 2'-deoxy-2'- fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring.
  • a modification of a sugar of a nucleotide may comprise a 2'-oxygen of a sugar is linked to a l'-carbon or 4'-carbon of the sugar, or a 2'-oxygen is linked to the l'-carbon or d'carbon via an ethylene or methylene bridge.
  • a modified nucleotide has an acyclic sugar that lacks a 2'-carbon to 3 '-carbon bond.
  • a modified nucleotide has a thiol group, e.g., in the 4' position of the sugar.
  • a lipid-conjugated RNAi oligonucleotide described herein comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more).
  • the sense strand of the lipid-conjugated RNAi oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more).
  • the antisense strand of the lipid-conjugated RNAi oligonucleotide comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).
  • all the nucleotides of the sense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the antisense strand of the lipid-conjugated RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the lipid-conjugated RNAi oligonucleotide (/. ⁇ ., both the sense strand and the antisense strand) are modified.
  • the modified nucleotide comprises a 2'-modification (e.g., a 2'-F or 2'-0Me, 2'-M0E, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid).
  • a 2'-modification e.g., a 2'-F or 2'-0Me, 2'-M0E, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid.
  • the disclosure provides lipid-conjugated RNAi oligonucleotides having different modification patterns.
  • the modified lipid-conjugated RNAi oligonucleotides comprise a sense strand sequence having a modification pattern as set forth in the Examples and Sequence Listing and an antisense strand having a modification pattern as set forth in the Examples and Sequence Listing.
  • a lipid-conjugated RNAi oligonucleotide disclosed herein comprises an antisense strand having nucleotides that are modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises an antisense strand comprises nucleotides that are modified with 2'-F and 2'-0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand having nucleotides that are modified with 2'-F.
  • a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand comprising nucleotides that are modified with 2'-F and 2'-0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide disclosed herein comprises a sense strand comprising nucleotides that are modified with 2'-F and 2'-OMe, provided that a nucleotide conjugated to a lipid moiety is not modified with 2’-F or 2’-0Me.
  • an oligonucleotide described herein comprises a sense strand with about 10-25%, 10%, 11%, 12%, 13%, 14% 15%, 16%, 17%, 18%, 19% or 20% of the nucleotides of the sense strand comprising a 2’ -fluoro modification.
  • about 11% of the nucleotides of the sense strand comprise a 2-fluoro modification.
  • about 20% of the nucleotides of the sense strand comprise a 2-fluoro modification.
  • an oligonucleotide described herein comprises an antisense strand with about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprising a 2’ -fluoro modification. In some embodiments, about 32% of the nucleotides of the antisense strand comprise a 2’ -fluoro modification. In some embodiments, the oligonucleotide has about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of its nucleotides comprising a 2’-fluoro modification.
  • nucleotides in the oligonucleotide comprise a 2’ -fluoro modification. In some embodiments, about 26% of the nucleotides in the oligonucleotide comprise a 2’ -fluoro modification.
  • one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group.
  • one or more nucleotides forming a base pair with a nucleotide at one or more of positions 10-13 of the antisense strand is modified with a 2'-F group.
  • the sugar moiety at each of nucleotides not modified with a 2’-F group or conjugated to a lipid in the sense strand is modified with a 2'-OMe.
  • the sugar moiety at each of nucleotides at positions 1-7 and 12-20 in the sense strand is modified with a 2'-OMe.
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand 22 nucleotides in length, with positions 1-22 numbered 5’ to 3’, and a sense strand having a 2’ -fluoro modification at each of the nucleotides forming a base pair with nucleotides at one or more of positions 10, 11, 12, and 13 of the antisense strand.
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand 22 nucleotides in length, with positions 1-22 numbered 5’ to 3’, and a sense strand having a 2’ -fluoro modification at each of the nucleotides forming a base pair with nucleotides at positions 10, 11, 12, 13, or any combination thereof, of the antisense strand.
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand 22 nucleotides in length, with positions 1-22 numbered 5’ to 3’, and a sense strand having a 2’ -fluoro modification at each of the nucleotides forming a base pair with nucleotides at positions 10, 11, 12, and 13 of the antisense strand.
  • the sense strand comprises at least one 2’-F modified nucleotide wherein the remaining nucleotides not modified with a 2’-F group or conjugated to a lipid are modified with a 2’-OMe.
  • the antisense strand has 7 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-F. In some embodiments, the sugar moiety at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with a 2’-F. In some embodiments, the antisense strand has 14 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-OMe. In some embodiments, the sugar moiety at positions 6, 8, 9, 11, 12, 13, 15, 16,
  • the sense strand has 4 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-F. In some embodiments, the sugar moiety at positions 2, 3, 8, 9, 10, and 11 of the sense strand are modified with a 2’-F. In some embodiments, the sense strand has 15 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-OMe. In some embodiments, the sugar moiety at positions 6, 8, 9, 11, 12, 13, 15, 16, 17,
  • the sense strand comprises a 2’ -fluoro modification at positions 3-6 or 4-7, numbered 5’ to 3’. In some embodiments, the sense strand comprises a 2’-fluoro modification at positions 3-6. In some embodiments, the sense strand comprises a 2’-fluoro modification at positions 4-7.
  • the antisense strand has 3 nucleotides that are modified at the 2'-position of the sugar moiety with a 2'-F.
  • the sugar moiety at positions 2, 5 and 14 and optionally up to 3 of the nucleotides at positions 1, 3, 7 and 10 of the antisense strand are modified with a 2'-F.
  • the sugar moiety at each of the positions at positions 2, 5 and 14 of the antisense strand is modified with the 2'-F.
  • the sugar moiety at each of the positions at positions 1, 2, 5 and 14 of the antisense strand is modified with the 2'-F.
  • the sugar moiety at each of the positions at positions 2, 4, 5 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 7 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 1, 2, 3, 5, 10 and 14 of the antisense strand is modified with the 2'-F.
  • the sugar moiety at each of the positions at positions 2, 3, 4, 5, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 5, 7, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand is modified with the 2'-F. In some embodiments, the antisense strand has 9 nucleotides that are modified at the 2'-position of the sugar moiety with a 2'-F. In some embodiments, the sugar moiety at each of the positions at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 of the antisense strand is modified with the 2'-F.
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 5, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2’-O-methyl (2'-0Me), 2’-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2’-deoxy-2’-fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'- aminoethyl (EA), 2’-O-methyl (2'-0Me), 2’-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2’-deoxy-2’-fluoro-P-d-arabinonucleic acid (2'- FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 5, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 1, 2, 3, 5, 7, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions
  • a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, 14, 16 and 19 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'- aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'- FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2 '-O-m ethoxy ethyl (2'-M0E), 2'-O-[2-(methylamino)-2- oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-F.
  • a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with 2'-0Me.
  • a lipid-conjugated RNAi oligonucleotide comprises an antisense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, or position 22 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'- 0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 8-11 modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 3, 5, 8, 10, 12, 13, 15 and 17 modified with 2'-F. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1-7 and 12-17 or 12-20 modified with 2’0Me.
  • a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 2-7 and 12-17 or 12-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1-6 and 12-17 or 12-20 modified with 2’0Me. In some embodiments, a lipid-conjugated RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1, 2, 4, 6, 7, 9, 11, 14, 16 and 18-20 modified with 2’0Me.
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O- methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O- NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 2-7 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O- propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O- methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'- fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1-6 and 12-17 or 12-20 of the sense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2- (methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'- FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at positions 1, 2, 4, 6, 7, 9, 11, 14, 16 and 18- 20 of the sense strand modified with a modification selected from the group consisting of 2'- O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy- 2'-fluoro-P-d-arabinonucleic acid (2'-FANA).
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-F.
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37 or position 38 modified with 2'-F.
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with 2'-0Me.
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37 or position 38 modified with 2'-0Me.
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, or position 36 modified with a modification selected from the group consisting of 2'-O- propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’ -aminoethyl (EA), 2'-O-methyl (2'-0Me), 2'- O-methoxy ethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'- fluoro-
  • a lipid-conjugated RNAi oligonucleotide comprises a sense strand having the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37 or position 38 modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2’-aminoethyl (EA), 2'-O- methyl (2'-0Me), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O- NMA), and 2'-deoxy-2'
  • a lipid-conjugated RNAi oligonucleotide described herein comprises a 5 ’-terminal phosphate.
  • the 5 '-terminal phosphate groups of the lipid-conjugated RNAi oligonucleotide enhance the interaction with Ago2.
  • oligonucleotides comprising a 5'-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo.
  • a lipid-conjugated RNAi oligonucleotide herein comprises analogs of 5' phosphates that are resistant to such degradation.
  • the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, or a combination thereof.
  • the 5' end of a lipid-conjugated RNAi oligonucleotide strand is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5'- phosphate group (“phosphate mimic”).
  • a lipid-conjugated RNAi oligonucleotide herein has a phosphate analog at a 4'-carbon position of the sugar (referred to as a “4'-phosphate analog”). See, e.g., Inti. Patent Application Publication No. WO 2018/045317.
  • a lipid- conjugated RNAi oligonucleotide herein comprises a 4'-phosphate analog at a 5 '-terminal nucleotide.
  • a phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety e.g., at its 4'-carbon) or analog thereof.
  • a 4'-phosphate analog is a thiomethyl phosphonate or an aminomethyl phosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the amino methyl group is bound to the 4'-carbon of the sugar moiety or analog thereof.
  • a 4'-phosphate analog is an oxymethyl phosphonate.
  • an oxymethyl phosphonate is represented by the formula -O-CH2-PO(OH)2,- O-CH2-PO(OR)2, or -O-CH2-POOH(R), in which R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si (CH3)3 or a protecting group.
  • the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3 or CH2CH3.
  • R is CH3.
  • the 4’- phosphate analog is 5’-methoxyphosphonate-4’-oxy. In some embodiments, the 4’ -phosphate analog is 4’ -oxymethyl phosphonate.
  • a lipid-conjugated RNAi oligonucleotide provided herein comprises an antisense strand comprising a 4'-phosphate analog at the 5 '-terminal nucleotide, wherein 5’-terminal nucleotide comprises the following structure:
  • a lipid-conjugated RNAi oligonucleotide herein comprises a modified internucleoside linkage.
  • phosphate modifications or substitutions result in an oligonucleotide that comprises at least about 1 (e.g., at least 1, at least 2, at least 3 or at least 5) modified internucleotide linkage.
  • any one of the oligonucleotides disclosed herein comprises about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified internucleotide linkages.
  • any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified internucleotide linkages.
  • a modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage.
  • at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage.
  • a lipid-conjugated RNAi oligonucleotide provided herein has a phosphorothioate linkage between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 18 and 19 of the sense strand, positions 19 and 20 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, the third to last position and penultimate position of the sense strand, and the penultimate position and ultimate position of the sense strand.
  • the oligonucleotide described herein has a phosphorothioate linkage between positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 13 and 14 of the antisense strand, positions 14 and 15 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide described herein has a phosphorothioate linkage between positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 16 and 17 of the antisense strand, positions 17 and 18 of the antisense strand, positions 18 and 19 of the antisense strand, positions 19 and 20 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide described herein has a phosphorothioate linkage between positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 13 and 14 of the antisense strand, positions 14 and 15 of the antisense strand, positions 16 and 17 of the antisense strand, positions 17 and 18 of the antisense strand, positions 18 and 19 of the antisense strand, positions 19 and 20 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide comprises phosphorothioate linkages on the sense strand between nucleotides at positions 1 and 2, 8 and 9, and 9 and 10. In some embodiments, the oligonucleotide comprises phosphorothioate linkages on the antisense strand between nucleotides at positions 1 and 2, 2 and 3, 3 and 4, 12 and 14, 14 and 15, 20 and 21, and 21 and 22.
  • the oligonucleotide described herein has a phosphorothioate linkage between positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 12 and 13 of the antisense strand, positions 13 and 14 of the antisense strand, positions 14 and 15 of the antisense strand, positions 15 and 16 of the antisense strand, positions 16 and 17 of the antisense strand, positions 17 and 18 of the antisense strand, positions 18 and 19 of the antisense strand, positions 19 and 20 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.
  • the oligonucleotide comprises a nucleotide at position 14 of a 22 nucleotide antisense strand, wherein the nucleotide is flanked by phosphorothioate linkages (i.e. a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15).
  • the flanked nucleotide at position 14 is the ultimate nucleotide of a duplex between the antisense strand and sense strand.
  • the oligonucleotide comprises a sense and antisense strand
  • the antisense strand comprises a flanked oligonucleotide at position 14 of a 22 nucleotide antisense strand (i.e. a phosphorothioate linkage between positions 13 and 14 and between positions 14 and 15), wherein the sense and antisense strand form a duplex and the antisense strand comprises an overhang, and wherein the nucleotide at position 14 is within the overhang.
  • an oligonucleotide conjugate described herein comprises a peptide nucleic acid (PNA).
  • PNAs are oligonucleotide mimics in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton, composed of N-(2- aminoethyl)glycine units. Nucleobases are linked to this skeleton through a two-atom carboxymethyl spacer.
  • an oligonucleotide conjugate described herein comprises a morpholino oligomer (PMO) comprising an intemucleotide linkage backbone of methylene morpholine rings linked through phosphorodiamidate groups.
  • PMO morpholino oligomer
  • a lipid-conjugated RNAi oligonucleotide herein comprises one or more modified nucleobases.
  • modified nucleobases also referred to herein as base analogs
  • a modified nucleobase is a nitrogenous base.
  • a modified nucleobase does not contain nitrogen atom. See, e.g., US Patent Application Publication No. 2008/0274462.
  • a modified nucleotide comprises a universal base.
  • a modified nucleotide does not contain a nucleobase (abasic).
  • a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering structure of the duplex.
  • a reference single-stranded nucleic acid e.g., oligonucleotide
  • a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower Tm than a duplex formed with the complementary nucleic acid.
  • the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid comprising the mismatched base.
  • Non-limiting examples of universal-binding nucleotides include, but are not limited to, inosine, l-P-D-ribofuranosyl-5-nitroindole and/or l-P-D-ribofuranosyl-3 -nitropyrrole (see, US Patent Application Publication No. 2007/0254362; Van Aerschot et al. (1995) NUCLEIC ACIDS RES. 23:4363-70; Loakes etal. (1995) NUCLEIC ACIDS RES. 23:2361-66; and Loakes & Brown (1994) NUCLEIC ACIDS RES. 22:4039-43).
  • the oligonucleotide described herein comprises at least one Tm- increasing nucleotide in the sense strand. In some embodiments, the oligonucleotide has one Tm-increasing nucleotide in the sense strand. In some embodiments, the oligonucleotide has up to two Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to three Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to four Tm-increasing nucleotides in the sense strand.
  • the oligonucleotide has up to five Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to six Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to seven Tm- increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to eight Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to nine Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to ten Tm-increasing nucleotides in the sense strand.
  • the oligonucleotide has 1 to 2 Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has 1 to 3 Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has 1 to 4 Tm-increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has 1 to 5 Tm- increasing nucleotides in the sense strand.
  • an oligonucleotide comprising a stem-loop comprises a Tm- increasing nucleotide in the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in at least one base pair of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in one base pair of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in two base pairs of the stem.
  • an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in three base pairs of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in four base pairs of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in five base pairs of the stem. In some embodiments, an oligonucleotide comprising a stem-loop comprises Tm-increasing nucleotides in six base pairs of the stem.
  • Tm-increasing nucleotides include, but are not limited to, bicyclic nucleotides, tricyclic nucleotides, a G-clamp, and analogues thereof, hexitol nucleotides, or a modified nucleotide.
  • the Tm-increasing nucleotide is a bicyclic nucleotide.
  • the Tm-increasing nucleotide is a locked nucleic acid (LNA).
  • the sense strand of the oligonucleotide comprises a Tm- increasing nucleotide at one or more of positions 2, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, and 19. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 2. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 9. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 10.
  • the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 11. In some embodiments, the sense strand of the oligonucleotide comprises a Tm- increasing nucleotide at position 12. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 14. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 15. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 16.
  • the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 18. In some embodiments, the sense strand of the oligonucleotide comprises a Tm-increasing nucleotide at position 19.
  • a 10-nucleotide sense strand comprises a Tm-increasing nucleotide at one or more of positions 2, 6 and 7. In some embodiments, a 10-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at positions 2. In some embodiments, a 10-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at positions 2 and 6.
  • a 10-nucleotide sense strand comprises a Tm-increasing nucleotide at positions 2 and 7.
  • a 10- nucleotide sense strand comprises a Tm-increasing nucleotide at 6 and 7.
  • a 10-nucleotide sense strand comprises a Tm-increasing nucleotide at positions 2, 6 and 7.
  • a 12-nucleotide sense strand comprises a Tm-increasing nucleotide at one or more of positions 2, 7, 8, 10, and 11.
  • a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2.
  • a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2 and position 7.
  • a 12-nucleotide sense strand comprises a Tm- increasing nucleotide at position 2, position 7, and position 8. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 7, position 8, and position 10. In some embodiments, a 12-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm- increasing nucleotide at position 2, position 7, positions, position 10, and position 11.
  • a 12-nucleotide sense strand comprises a Tm-increasing nucleotide at position 2, position 10, and position 11.
  • the sense strand comprises a Tm-increasing nucleotide at position 2, position 11, and position 12.
  • a 14-nucleotide sense strand comprises a Tm-increasing nucleotide at one or more of positions 2, 9, 10, 12, and 13.
  • a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2.
  • a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2 and position 9.
  • a 14-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2, position 9, and position
  • a 14-nucleotide sense strand comprises a Tm- increasing nucleotide at position 2, position 9, position 10, position 12, and position 13.
  • a 16-nucleotide sense strand comprises a Tm-increasing nucleotide at one or more of positions 2, 11, 12, 14, and 15.
  • a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2.
  • a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2 and position 11.
  • a 16-nucleotide sense strand, with nucleotides numbered 5’ to 3’ comprises a Tm- increasing nucleotide at position 2, position
  • a 16-nucleotide sense strand comprises a Tm- increasing nucleotide at position 2, position 11, position
  • a 16-nucleotide sense strand comprises a Tm- increasing nucleotide at position 2, position 11, position 12, position 14., and position 15.
  • a 20-nucleotide sense strand comprises a Tm-increasing nucleotide at one or more of positions 2, 15, 16, 18, and 19. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2 and position 15. In some embodiments, a 20-nucleotide sense strand, with nucleotides numbered 5’ to 3’, comprises a Tm-increasing nucleotide at position 2, position
  • a 20-nucleotide sense strand comprises a Tm-increasing nucleotide at position 2, position 15, position
  • a 20-nucleotide sense strand comprises a Tm-increasing nucleotide at position 2, position 15, position 16, position 18, and position 19.
  • the disclosure provides an RNAi oligonucleotide for reducing target gene expression by the RNAi pathway comprising a combination of one or more Tm- increasing nucleotides and one or more nucleotides (e.g., a modified nucleotide) having a lower binding affinity, wherein the duplex region comprising the RNAi oligonucleotide is maintained under physiological conditions and the ability of the RNAi oligonucleotide to inhibit or reduce target gene expression is maintained.
  • Bicyclic nucleotides typically have a sugar moiety with a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
  • Such bicyclic nucleotides have various names including BNA's and LNA's for bicyclic nucleic acids and locked nucleic acids, respectively.
  • the synthesis of bicyclic nucleotides and their incorporation into nucleic acid compounds has also been reported in the literature, including, for example, Singh et al., Chem.
  • the Tm-increasing nucleotide is a bicyclic nucleotide that comprises a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a first ring of 4 to 7 members and a bridge forming a North-type sugar confirmation that connects any two atoms of the first ring of the sugar moiety to form a second ring.
  • the bridge connects the 2'-carbon and the 4'-carbon of the first ring to form a second ring.
  • the bridge contains 2 to 8 atoms. In certain embodiments, the bridge contains 3 atoms. In certain embodiments, the bridge contains 4 atoms. In certain embodiments, the bridge contains 5 atoms. In certain embodiments, the bridge contains 6 atoms. In certain embodiments, the bridge contains 7 atoms. In certain embodiments, the bridge contains 8 atoms. In certain embodiments, the bridge contains more than 8 atoms.
  • the bicyclic sugar moiety is a substituted furanosyl comprising a bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl to form the second ring.
  • the bicyclic nucleotide has the structure of Formula I: Formula I wherein B is a nucleobase; wherein G is H, OH, NH2, Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-Ce alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio; wherein X is O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; and wherein Wa and Wb are each independently, H, OH, a hydroxyl
  • G is H and X is NRi, wherein Ri is benzene or pyrene. In certain embodiments, of Formula I, G is H and X is S.
  • G is H and X is O:
  • G is H and X is NRi, wherein Ri is H, CH3, or
  • G is OH or NH2 and X is O.
  • G is OH and X is O:
  • G is NH2 and X is O:
  • G is CH3 or CH2OCH3 and X is O. In certain embodiments, of Formula I, G is CH3 and X is O : Formula le
  • G is CH2OCH3 and X is O:
  • the bicyclic nucleotide has the structure of Formula II: Formula II wherein B is a nucleobase; wherein Qi is CH2 or O; wherein X is CH2, O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein if Qi is O, X is CH2; wherein if Qi is CH2, X is CH2, O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula II to another nucleotide or to an oligonucleotide and wherein at least one of Wa or
  • Wb is an intemucleotide linking group attaching the nucleotide represented by Formula II to an oligonucleotide.
  • Qi is CH2 and X is NRi, wherein Ri is H, CH3 or OCH3: Formula lie
  • Qi is CH2 and X is NH:
  • the bicyclic nucleotide has the structure of Formula III: Formula III wherein B is a nucleobase; wherein Q2 is O or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein X is CH2, O, S, or NRi, wherein Ri is H, Ci-Ce alkyl, Ci-Ce alkoxy, benzene or pyrene; wherein if Q2 is O, X is NRi; wherein if Q2 is NRi, X is O or S; wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula III to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an intemucleotide linking group attaching the
  • Q2 is O and X is NRi. In certain embodiments of Formula III, Q2 is O and X is NRi, wherein Ri is Ci-Ce alkyl. In certain embodiments of Formula III, Q2 is O and X is NRi and Ri is H or CH3
  • Q2 is O and X is NRi and Ri is CH3:
  • Q2 is NRi and X is O. In certain embodiments of Formula III, Q2 is NRi, wherein Ri is Ci-Ce alkyl and X is O.
  • Q2 is NCH3 and X is O: Formula Illb
  • the bicyclic nucleotide has the structure of Formula IV: Formula IV wherein B is a nucleobase; wherein Pi and P3 are CH2, P2 is CH2 or O and P4 is O; and wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the nucleotide represented by Formula IV to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an internucleotide linking group attaching the nucleotide represented by Formula IV to an oligonucleotide.
  • Pi and P3 are CH2, P2 is O and P4 is O: Formula IVb
  • the bicyclic nucleotide has the structure of Formula Va or Vb:
  • the bicyclic sugar moiety is a substituted furanosyl comprising a bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl to form the second ring, wherein the bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl includes, but is not limited to: a) 4'-CH 2 -O-N(R)-2' and 4'-CH 2 -N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group, including, for example, 4'-CH2-NH-O-2' (also known as BNA NC ), 4'-CH 2 -N(CH 3 )-O-2' (also known as BNA NC [NMe]), (as described in U.S.
  • Patent No. 7,427,672 which is hereby incorporated by reference in its entirety); b) 4'-CH 2 -2'; 4'-(CH 2 )2-2'; 4'-(CH 2 ) 3 -2'; 4'-(CH 2 )-O-2' (also known as LNA); 4'-(CH 2 )-S-2'; 4'-(CH 2 )2-O-2' (also known as ENA); 4'-CH(CH 3 )-O-2' (also known as cEt); and 4'-CH(CH2OCH3)-O-2' (also known as cMOE), and analogs thereof (as described in U.S. Patent No.
  • the bicyclic nucleotide (BN) is one or more of the following: (a) methyleneoxy BN, (b) ethyleneoxy BN, (c) aminooxy BN; (d) oxyamino BN, (e) methyl(methyleneoxy) BN (also known as constrained ethyl or cET), (f) methylene-thio BN, (g) methylene amino BN, (h) methyl carbocyclic BN, and (i) propylene carbocyclic BN, as shown below.
  • B is a nucleobase
  • R2 is H or CH3
  • Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an intemucleotide linking group attaching the bicyclic nucleotide to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an intemucleotide linking group attaching the bicyclic nucleotide to an oligonucleotide.
  • R2 is CH3, as follows (also known as
  • bicyclic sugar moieties and bicyclic nucleotides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • the bicyclic sugar moiety or nucleotide is in the a-L configuration.
  • the bicyclic sugar moiety or nucleotide is in the P-D configuration.
  • the bicyclic sugar moiety or nucleotide comprises a 2'0,4'-C- methylene bridge (2'-O-CH2-4') in the a-L configuration (a-L LNA).
  • the bicyclic sugar moiety or nucleotide is in the R configuration.
  • the bicyclic sugar moiety or nucleotide is in the S configuration.
  • the bicyclic sugar moiety or nucleotide comprises a 4'-CH(CH3)-O-2' bridge (i.e., cEt) in the S-configuration.
  • the Tm-increasing nucleotide is a tricyclic nucleotide.
  • the synthesis of tricyclic nucleotides and their incorporation into nucleic acid compounds has also been reported in the literature, including, for example, Steffens et al., J. AM. CHEM. SOC. 1997;119: 11548-549; Steffens et al., J. ORG. CHEM. 1999;121(14):3249-55; Renneberg et al., J. AM. CHEM. SOC. 2002;124:5993-6002; Ittig et al., NUCLEIC ACIDS RES.
  • the tricyclic nucleotide is a tricyclo nucleotide (also called tri cyclo DNA) in which the 3 '-carbon and 5 '-carbon centers are connected by an ethylene that is fused to a cyclopropane ring, as discussed for example in Leumann CJ, BIOORG. MED. CHEM. 2002;10:841-54 and published U.S. Applications 2015/0259681 and 2018/0162897, which are each hereby incorporated by reference.
  • tricyclo nucleotide also called tri cyclo DNA
  • the tricyclic nucleotide comprises a substituted furanosyl ring comprising a bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl to form a second ring, and a third fused ring resulting from a group connecting the 5 '-carbon to the methylene group of the bridge that connects the 2'-carbon and the 4'-carbon of the furanosyl, as discussed, for example, in published U.S. Application 2015/0112055, which is hereby incorporated by reference.
  • the Tm-increasing nucleotide is a G-clamp, guanidine G-clamp or analogue thereof (Wilds et al., CHEM, 2002; 114: 123 and Wilds et al., CHIM ACTA 2003; 114: 123), a hexitol nucleotide (Herdewijn, CHEM. BIODIVERSITY 2010;7: 1-59), or a modified nucleotide.
  • the modified nucleotide can have a modified nucleobase, as described herein, including for example, 5- bromo-uracil, 5-iodo-uracil, 5-propynyl-modified pyrimidines, or 2-amino adenine (also called 2,6-diaminopurine) (Deleavey et al., CHEM. & BIOL. 2012;19:937-54) or 2-thio uridine, 5 Me- thio uridine, and pseudo uridine.
  • the modified nucleotide can also have a modified sugar moiety, as described for example, in U.S. Patent No.
  • the Tm-increasing nucleotide is a bicyclic nucleotide. In certain embodiments, the Tm-increasing nucleotide is a tricyclic nucleotide. In certain embodiments, the Tm-increasing nucleotide a G-clamp, guanidine G-clamp or analogue thereof. In certain embodiments, the Tm-increasing nucleotide is a hexitol nucleotide. In certain embodiments, the Tm-increasing nucleotide is a bicyclic or tricyclic nucleotide.
  • the Tm- increasing nucleotide is a bicyclic nucleotide, a tricyclic nucleotide, or a G-clamp, guanidine G-clamp or analogue thereof.
  • the Tm-increasing nucleotide is a bicyclic nucleotide, a tricyclic nucleotide, a G-clamp, guanidine G-clamp or analogue thereof, or a hexitol nucleotide.
  • the Tm-increasing nucleotide increases the T m of the nucleic acid inhibitor molecule by at least 2 °C per incorporation. In certain embodiments, the Tm- increasing nucleotide increases the Tm of nucleic acid inhibitor molecule by at least 3 °C per incorporation. In certain embodiments, the Tm-increasing nucleotide increases the Tm of nucleic acid inhibitor molecule by at least 4 °C per incorporation. In certain embodiments, the Tm-increasing nucleotide increases the Tm of nucleic acid inhibitor molecule by at least 5 °C per incorporation.
  • oligonucleotides of the disclosure e.g., lipid-conjugated RNAi oligonucleotides
  • CNS central nervous system
  • a lipid-conjugated RNAi oligonucleotide disclosed herein is modified to facilitate targeting and/or delivery to a particular tissue, cell, or organ (e.g., to facilitate delivery of the conjugate to the CNS).
  • a lipid- conjugated RNAi oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more targeting ligand(s).
  • nucleotides of a lipid- conjugated RNAi oligonucleotide disclosed herein are each conjugated to a separate targeting ligand.
  • 1 nucleotide of a lipid-conjugated RNAi oligonucleotide herein is conjugated to a separate targeting ligand.
  • 2 to 4 nucleotides of a lipid- conjugated RNAi oligonucleotide herein are each conjugated to a separate targeting ligand.
  • targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., targeting ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5' or 3' end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the lipid-conjugated RNAi oligonucleotide resembles a toothbrush.
  • a lipid-conjugated RNAi oligonucleotide may comprise a stem-loop at either the 5' or 3' end of the sense strand and 1, 2, 3 or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand.
  • a lipid-conjugated RNAi oligonucleotide provided by the disclosure comprises a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a triloop or a tetraloop, and wherein the 3 or 4 nucleotides comprising the triloop or tetraloop, respectfully, are individually conjugated to a targeting ligand.
  • GalNAc is a high affinity ligand for the ASGPR, which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalizing and subsequent clearing circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotide of the instant disclosure can be used to target these oligonucleotides to the ASGPR expressed on cells.
  • an oligonucleotide of the instant disclosure is conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotide to an ASGPR expressed on human liver cells (e.g., human hepatocytes).
  • the GalNAc moiety target the oligonucleotide to the liver.
  • an oligonucleotide of the disclosure is conjugated directly or indirectly to a monovalent GalNAc.
  • the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (i.e., is conjugated to 2, 3 or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties).
  • an oligonucleotide is conjugated to one or more bivalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties.
  • nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety.
  • 2 to 4 nucleotides of a tetraloop are each conjugated to a separate GalNAc.
  • 1 to 3 nucleotides of a triloop are each conjugated to a separate GalNAc.
  • targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5' or 3' end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush.
  • GalNAc moieties are conjugated to a nucleotide of the sense strand.
  • four (4) GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand where each GalNAc moiety is conjugated to 1 nucleotide.
  • the tetraloop is any combination of adenine and guanine nucleotides. In some embodiments, the tetraloop is any combination of adenine, guanine, cytosine, and uridine nucleotides.
  • a lipid-conjugated RNAi oligonucleotide herein comprises a monovalent GalNAc attached to a guanine nucleotide referred to as [ademG-GalNAc] or 2'- aminodiethoxymethanol-Guanine-GalNAc, as depicted below:
  • a lipid-conjugated RNAi oligonucleotide herein comprises a monovalent GalNAc attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'- aminodiethoxymethanol-Adenine-GalNAc, as depicted below:
  • a targeting ligand is conjugated to a nucleotide using a click linker.
  • an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal- based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016/100401.
  • the linker is a labile linker. However, in other embodiments, the linker is stable.
  • a loop comprising from 5' to 3' the nucleotides GAAA, in which GalNAc moi eties are attached to nucleotides of the loop using an acetal linker.
  • Such a loop may be present, for example, at positions 27-30 of the sense strand.
  • a targeting ligand is conjugated to a nucleotide using a click linker.
  • an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016/100401.
  • the linker is a labile linker. However, in other embodiments, the linker is a stable linker.
  • a duplex extension (e.g., of up to 3, 4, 5 or 6 bp in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a lipid-conjugated RNAi oligonucleotide.
  • a targeting ligand e.g., a GalNAc moiety
  • a lipid-conjugated RNAi oligonucleotide herein does not have a GalNAc conjugated thereto.
  • any of the lipid moieties described herein are conjugated to a nucleotide of the sense strand of the oligonucleotide.
  • a lipid moiety is conjugated to a terminal position of the oligonucleotide.
  • the lipid moiety is conjugated to the 5’ terminal nucleotide of the sense strand.
  • the lipid moiety is conjugated to the 3’ terminal nucleotide of the sense strand.
  • the lipid moiety is conjugated to an internal nucleotide on the sense strand.
  • An internal position is any nucleotide position other than the two terminal positions from each end of the sense strand.
  • the lipid moiety is conjugated to one or more internal positions of the sense strand. In some embodiments, the lipid moiety is conjugated to position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position
  • the lipid moiety is conjugated to position 1 of the sense strand, In some embodiments, the lipid moiety is conjugated to position 2 of the sense strand, In some embodiments, the lipid moiety is conjugated to position 4 of the sense strand, In some embodiments, the lipid moiety is conjugated to position 6 of the sense strand, In some embodiments, the lipid moiety is conjugated to position 8 of the sense strand, In some embodiments, the lipid moiety is conjugated to position 15 of the sense strand, In some embodiments, the lipid moiety is conjugated to position 28 of the sense strand. In some embodiments, the lipid moiety is conjugated to position 38 of the sense strand.
  • the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 20, position 19, position 18, position 17, position 16, position 15, position 14, position 13, or position 12 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 16, position 14, or position 12 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 20 of the antisense strand.
  • the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 19 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 18 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 17 of the antisense strand.
  • the lipid moiety is conjugated to a nucleotide of the sense strand that forms abase pair with a nucleotide at position 16 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 15 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 14 of the antisense strand.
  • the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 13 of the antisense strand. In some embodiments, the lipid moiety is conjugated to a nucleotide of the sense strand that forms a base pair with a nucleotide at position 12 of the antisense strand.
  • a lipid-conjugated RNAi oligonucleotide described herein comprises at least one nucleotide conjugated with one or more lipid moieties.
  • the one or more lipid moieties are conjugated to the same nucleotide.
  • the one or more lipid moieties are conjugated to different nucleotides.
  • one, two, three, four, five, or six lipid moieties are conjugated to the oligonucleotide.
  • one or more lipid moieties are conjugated to an adenine nucleotide.
  • one or more lipid moieties are conjugated to a guanine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a cytosine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a thymine nucleotide. In some embodiments, one or more lipid moieties are conjugated to a uracil nucleotide.
  • the lipid moiety is a hydrocarbon chain. In some embodiments, the hydrocarbon chain is saturated. In some embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the hydrocarbon chain is branched. In some embodiments, the hydrocarbon chain is straight. In some embodiments, the lipid moiety is a C8-C30 hydrocarbon chain.
  • the lipid moiety is a C8:0, C10:0, Cl 1 :0, C12:0, C14:0, C16:0, C17:0, C18:0, C18:l, C18:2, C22:5, C22:0, C24:0, C26:0, C22:6, C24:l, diacyl C16:0 or diacyl C18: l.
  • the lipid moiety is a C 16 hydrocarbon chain.
  • the lipid moiety is conjugated to the oligonucleotide via a linker.
  • a nucleotide of the lipid-conjugated oligonucleotide is represented by formula Il-b or II-c:
  • L 1 is a covalent bond, a monovalent or a bivalent saturated or unsaturated, straight, or branched Ci-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -
  • R 4 is hydrogen, R A , or a suitable amine protection group
  • R 5 is adamantyl, or a saturated or unsaturated, straight, or branched C1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) 2 -, -P(O)OR-, or -P(S)OR.
  • R 5 is selected from
  • lipid-conjugated RNAi oligonucleotide In certain embodiments of the lipid-conjugated RNAi oligonucleotide,
  • R 5 is selected from
  • R 5 is
  • a nucleotide of the lipid-conjugated RNAi oligonucleotide is represented by formula Il-Ib or II-Ic:
  • B is a nucleobase or hydrogen; m is 1-50;
  • X 1 is -O-, or -S-;
  • Y is hydrogen
  • R 3 is hydrogen, or a suitable protecting group
  • X 2 is O, or S
  • X 3 is -O-, -S-, or a covalent bond
  • Y 1 is a linking group attaching to the 2'- or 3 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide;
  • Y 2 is hydrogen, a phosphoramidite analogue, an internucleotide linking group attaching to the 5 '-terminal of a nucleoside, a nucleotide, or an oligonucleotide, or a linking group attaching to a solid support;
  • R 5 is adamantyl, or a saturated or unsaturated, straight, or branched C1-50 hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by - O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O) 2 -, -P(O)OR-, or -P(S)OR-; and R is hydrogen, a suitable protecting group, or an optionally substituted group selected from Ci- 6 aliphatic, phenyl, a 4-7 membered saturated or partially unsaturated heterocyclic having 1-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • the lipid is In some embodiments, the oligonucleotide of the oligonucleotide-ligand conjugate is a double-stranded molecule. In some embodiments, the oligonucleotide is an RNAi molecule. In some embodiments, the double stranded oligonucleotide comprises a stem loop. In some embodiments, the stem loop is set forth as S1-L-S2, wherein SI is complementary to S2, and wherein L forms a loop between SI and S2. In some embodiments, the ligand is conjugated to any of the nucleotides in the loop of the stem loop.
  • the ligand is conjugated to any of the nucleotides in the stem of the stem loop. In some embodiments, the ligand is conjugated to the first nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the second nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the third nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to the fourth nucleotide from 5’ to 3’ in the loop. In some embodiments, the ligand is conjugated to one, two, three, or four of the nucleotides in the loop. In some embodiments, the ligand is conjugated to three of the nucleotides in the stem loop.
  • the stem loop is 16 nucleotides in length.
  • the ligand is conjugated to the third nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the eighth nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the ninth nucleotide from 5’ to 3’ in the stem loop. In some embodiments, the ligand is conjugated to the tenth nucleotide from 5’ to 3’ in the stem loop.
  • the lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a fatty acid.
  • the fatty acid is a saturated fatty acid.
  • the fatty acid is an unsaturated fatty acid.
  • lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a lipid.
  • the lipid is a carbon chain. In some embodiments, the carbon chain is saturated. In some embodiments, the carbon chain is unsaturated.
  • the lipid- conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a 16-carbon (Cl 6) lipid. In some embodiments, the C16 lipid comprises at least one double bond. In some embodiments, the lipid-conjugated RNAi oligonucleotide comprises a nucleotide conjugated with a 22-carbon (C22) lipid. In some embodiments, the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C16 lipid as shown in:
  • the oligonucleotide of the lipid-conjugated RNAi oligonucleotide is conjugated to a C22 lipid as shown in:
  • the 3’ end of the sense strand is a blunt end. In some embodiments, the 5’ end of the antisense strand is a blunt end. In some embodiments, the 3’ end of the antisense strand comprises an overhang. In some embodiments, the 5’ end of the antisense strand comprises an overhang. In some embodiments, the 5’ and 3’ ends of the antisense strand each comprise an overhang.
  • the lipid-conjugated RNAi oligonucleotide comprises one or more 2’ modifications.
  • the 2’ modifications are selected from 2’-fluoro and 2 ’-methyl.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [mXs][mXs][mX][mX][mX][ademX-L][mX][mX][mX][mX][mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][ademX-
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][mX][mX][ademX- L] [mX] [mX] [mX] [mX] [mX] [+X] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [m
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-Ls] [mX][mX][mX][mX][mX][mX][mX][fX][fX][fX][mX] [mX] [mX][mX][mXs][mX][mX] -3’ Hybridized to:
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-Ls] [+X][mX][mX][mX][mX][mX][fX][fX][fX][mX][mX] [mXs][+Xs][+X] -3’ Hybridized to:
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-Ls][mX][mX][mX][mX][mX][mX][mX][mX][mX][fX][fX][fX][mX][mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [mX] [
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][ademX-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+X][mX][mX][mX][mX][ademX- C 16] [mX] [mX] [mX] [mX] [mX] [+X] [mX]
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • Antisense Strand 5’ - [MePhosphonate-4O- mXs] [fXs] [fX] [fX] [mX] [fX] [mX] [mX] [fX] [fX]
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+Xs][ademX-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [+Xs][ademX- C22] [fX] [fX] [fX] [fX] [mX] [mX] [mX] [+X] [+Xs] [mXs] [mX] - 3 ’ Hybridized to:
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademX-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’-
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of Sense Strand: 5’- [ademXs-L] [mX/+X][mX][fX][mX][mX][mX][mX/+X] [mX/+X] [mX] [mXs/+Xs] [mX]-3 ’ Hybridized to:
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern of
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 2-14 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in Compound 15 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 16-18 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 19-38 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 39-40, 45, and 48-49 as described herein. In some embodiments, a lipid- conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 41-44 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 46-47 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 57 and 98-108 as described herein. In some embodiments, a lipid- conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 120-122 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 59-64 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 65-69 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 70-71 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 72-74 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 75-76 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 77-79 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in Compound 58 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 80-81 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 82-84 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 85-86 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 87-89 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 90-91 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 92-94 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in Compound 95 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 96-97 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 109-117 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 123-126 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of 127-130 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 131-136 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 137-140 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 141-145 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 146-148 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 149-154 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 155-160 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in Compound 161 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 162-165 and 171 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 166-170 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in Compound 172 as described herein.
  • a lipid- conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 173-176 and 182 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 177-181 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of 183-190 as described herein.
  • a lipid- conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 191-194 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 195-197 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 200-201 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 202-205, 207-210, and 211-215 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 217-218 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 219-222 and 224-232 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of 277-278 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 279-285 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 198-199 and 233 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 234-236 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 237-239 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 240-242 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 243-245 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 246-251 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 252-255 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in Compound 256 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 257-259 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 260-262 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 152 and 264-265 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 266-268 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 269-272 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 273-276 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 286 and 292 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 287-291 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 293-294 as described herein. In some embodiments, a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 2-15, 46 and 47 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 137-140. 146, 148, 162-165, 173-176, 183-190, 200, 217, 247, 270, 277, 278, 286 and 292 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 16, 19-28, 30-38, 41-44, 65-79, 80-97, 127-130, 202-205, 207-210, 212-215, 219-221, 224-227, 229-232, 237-239, 243-245, 252-255, 260-262, 266- 268, 274-276, 280-285, 287-291, and 293-294 as described herein.
  • a lipid-conjugated RNAi oligonucleotide for reducing expression of a target gene comprises the modification pattern as set forth in any one of Compounds 40, 45, 49, 59-64, 98, 99-117, 131- 136, 141-145, 149-160, 166-170, 177-181, 191-197, 201, 211, 218, 228, 234-236, 240-242, 248-251, 257-259, 264, 265, 272-273, and 279 as described herein.
  • reference to Compound numbers refers to the modification pattern (e.g., phosphorothioate linkages, 2’ modifications, conjugation) and not the nucleotide sequences.
  • nucleic acids and analogues thereof comprising lipid conjugate described herein can be made using a variety of synthetic methods known in the art, including standard phosphoramidite methods. Any phosphoramidite synthesis method can be used to synthesize the provided nucleic acids of this disclosure. In certain embodiments, phosphoramidites are used in a solid phase synthesis method to yield reactive intermediate phosphite compounds, which are subsequently oxidized using known methods to produce phosphonate-modified oligonucleotides, typically with a phosphodiester or phosphorothioate internucleotide linkages.
  • the oligonucleotide synthesis of the present disclosure can be performed in either direction: from 5' to 3' or from 3' to 5' using art known methods.
  • the method for synthesizing a provided nucleic acid comprises (a) attaching a nucleoside or analogue thereof to a solid support via a covalent linkage; (b) coupling a nucleoside phosphoramidite or analogue thereof to a reactive hydroxyl group on the nucleoside or analogue thereof of step (a) to form an intemucleotide bond there between, wherein any uncoupled nucleoside or analogue thereof on the solid support is capped with a capping reagent; (c) oxidizing said internucleotide bond with an oxidizing agent; and (d) repeating steps (b) to (c) iteratively with subsequent nucleoside phosphoramidites or analogue thereof to form a nucleic acid or analogue thereof, wherein at least the nucleoside or analogue thereof of step (a), the nucleoside phosphoramidite or analogue thereof of step (b) or at least one of the subsequent
  • an oligonucleotide is prepared comprising 1-3 nucleic acid or analogues thereof comprising lipid conjugates units on a tetraloop.
  • nucleic acids, and analogues thereof of the present disclosure are generally prepared according to Scheme A, Scheme Al and Scheme B set forth below:
  • a nucleic acid or analogue thereof of formula 1-1 is conjugated with one or more ligand/lipophilic compound to form a compound of formula I or la comprising one more ligand/lipid conjugates.
  • conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula 1-1 or I-la and one or more adamantyl and/or lipophilic compound (e.g., fatty acid) in series or in parallel by known techniques in the art.
  • nucleic acid or analogue thereof of formula I or la can then be deprotected to form a compound of formula 1-2 or I-2a and protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula 1-3 or I-3a.
  • a suitable hydroxyl protecting group e.g., DMTr
  • nucleic acid-ligand conjugates of formula 1-3 or I-3a can be covalently attached to a solid support (e.g., through a succinic acid linking group) to form a solid support nucleic acid-ligand conjugate or analogue thereof of formula 1-4 or I-4a comprising one or more adamantyl and/or lipid conjugate.
  • a nucleic acidligand conjugates of formula 1-3 or I-3a can react with a P(III) forming reagent (e.g., 2- cyanoethyl A,A-di-isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula 1-5 or I-5a comprising a P(III) group.
  • a nucleic acid-ligand conjugate or analogue thereof of formula 1-5 or I-5a can then be subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula 1-5 or I-5a is coupled to a solid supported nucleic acid-ligand conjugate or analogue thereof bearing a 5 ’-hydroxyl group.
  • Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, including one or more lipid conjugate nucleotide units represented by a compound of formula II-l or Il-Ia.
  • Each of B, E, L, ligand, LC, n, PG 1 , PG 2 , PG 4 , R 1 , R 2 , R 3 , X, X 1 , X 2 , X 3 , and Z is as defined above and described herein.
  • a nucleic acid or analogue thereof of formula 1-1 can be deprotected to form a compound of formula 1-6, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula 1-7, and reacted with a P(III) forming reagent (e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula 1-8 comprising a P(III) group.
  • a suitable hydroxyl protecting group e.g., DMTr
  • P(III) forming reagent e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite
  • a nucleic acid or analogue thereof of formula 1-8 is subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula 1-8 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
  • Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths represented by a compound of formula II-2.
  • An oligonucleotide of formula II-2 can then be conjugated with one or more ligands e.g., adamantyl, or lipophilic compound (e.g, fatty acid) to form a compound of formula II-l comprising one or more ligand conjugates.
  • ligands e.g., adamantyl, or lipophilic compound (e.g, fatty acid)
  • conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula II-2 and one or more adamantyl or fatty acid in series or in parallel by known techniques in the art.
  • Each of B, E, L, ligand, LC, n, PG 1 , PG 2 , PG 4 , R 1 , R 2 , R 3 , X, X 1 , X 2 , X 3 , and Z is as defined above and described herein.
  • nucleic acids, and analogues thereof of the present disclosure are prepared according to Scheme C and Scheme D set forth below:
  • nucleic acid or analogue thereof of formula Cl is protected to form a compound of formula C2.
  • Nucleic acid or analogue thereof of formula C2 is then alkylated (e.g., using DMSO and acetic acid via the Pummerer rearrangement) to form a monothioacetal compound of formula C3.
  • nucleic acid or analogue thereof of formula C3 is coupled with C4 under appropriate conditions (e.g., mild oxidizing conditions) to form a nucleic acid or analogue thereof of formula C5.
  • Nucleic acid or analogue thereof of formula C5 can then be deprotected to form a compound of formula C6 and coupled with a ligand (adamantyl or lipophilic compound (e.g., a fatty acid)) of formula C7 under appropriate amide forming conditions e.g., HATU, DIPEA), to form a nucleic acid-ligand conjugate or analogue thereof of formula I-b comprising a lipid conjugate of the disclosure.
  • Nucleic acid-ligand conjugate or analogue thereof of formula I-b can then be deprotected to form a compound of formula C8 and protected with a suitable hydroxyl protecting group e.g., DMTr) to form a compound of formula C9.
  • nucleic acid, or analogue thereof of formula C9 can be covalently attached to a solid support e.g., through a succinic acid linking group) to form a solid support nucleic acid-ligand conjugate or analogue thereof of formula CIO comprising a ligand conjugate (adamantyl or lipid moiety) of the disclosure.
  • a nucleic acid-ligand conjugate or analogue thereof of formula C9 can reacted with a P(III) forming reagent e.g., 2-cyanoethyl A,A-di-isopropylchlorophosphoramidite) to form a nucleic acidligand conjugate or analogue thereof of formula Cll comprising a P(III) group.
  • a nucleic acidligand conjugate or analogue thereof of formula Cll can then be subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula Cll is coupled to a solid supported nucleic acid-ligand conjugate or analogue thereof bearing a 5 ’-hydroxyl group.
  • Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, including one or more adamantyl and/or lipid conjugate nucleotide units represented by a compound of formula II-b-3.
  • Each of B, E, L 2 , PG 1 , PG 2 , PG 3 , PG 4 , R 1 , R 2 , R 3 , R 4 , R 5 , X 1 , X 2 , X 3 , V, W, and Z is as defined above and described herein.
  • Each of B, E, L 2 , PG 1 , PG 2 , PG 3 , PG 4 , R 1 , R 2 , R 3 , R 4 , R 5 , X 1 , X 2 , X 3 , V, W, and Z is as defined above and described herein.
  • a nucleic acid or analogue thereof of formula C5 can be selectively deprotected to form a compound of formula DI, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula D2, and reacted with a P(III) forming reagent (e.g., 2-cyanoethyl A,A-di- isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula D3.
  • a nucleic acid or analogue thereof of formula D3 is subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula D3 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group. Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and/or cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula D4.
  • An oligonucleotide of formula D4 can then be deprotected to form a compound of formula D5 and coupled with a hydrophobic ligand (e.g., adamantyl or a lipophilic moiety) to form a compound of formula C7 (e.g., adamantyl or a fatty acid) under appropriate amide forming conditions (e.g., HATU, DIPEA), to form an oligonucleotide of formula II-b-3 comprising a ligand (e.g., adamantyl or a fatty acid) conjugate of the disclosure.
  • a hydrophobic ligand e.g., adamantyl or a lipophilic moiety
  • C7 e.g., adamantyl or a fatty acid
  • appropriate amide forming conditions e.g., HATU, DIPEA
  • nucleic acid or analogues thereof of the disclosure such as aliphatic groups, alcohols, carboxylic acids, esters, amides, aldehydes, halogens, and nitriles can be interconverted by techniques well known in the art including, but not limited to reduction, oxidation, esterification, hydrolysis, partial oxidation, partial reduction, halogenation, dehydration, partial hydration, and hydration. See for example, “MARCH’S ADVANCED ORGANIC CHEMISTRY”, (5 th Ed., Ed.: Smith, M.B.
  • the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, said lipid conjugate unit represent by formula II-a-1: or a pharmaceutically acceptable salt thereof, comprising the steps of:
  • oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula I-5a is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
  • Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula Il-la comprising a lipid conjugate of the disclosure.
  • the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula I-5a: or a salt thereof, comprising the steps of:
  • nucleic acid or analogue thereof of formula I-3a with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula I-5a, wherein each of B, E, L, LC, n, PG 4 , R 1 , R 2 , R 3 , X, X 1 , X 2 , X 3 , E, and Z is as defined above and described herein.
  • PG 1 and PG 2 of a compound of formula la comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
  • reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A-butylammonium fluoride, and the like.
  • a compound of formula I-2a is protected with a suitable hydroxyl protecting group.
  • the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula I-2a includes an acid labile protecting group such as trityl, 4-methy oxytrityl, 4,4’ -dimethy oxytrityl, 4,4’,4”-trimethyoxytrityl, 9-phenyl- xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
  • the acid labile protecting group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.
  • a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
  • the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
  • the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
  • step (d) above is preformed using A,A-dimethylphosphoramic dichloride as a P(V) forming reagent.
  • the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugates, further comprising preparing a nucleic acid-lipid conjugate or analogue thereof of formula la: or a salt thereof, comprising the steps of:
  • a nucleic acid or analogue thereof of formula I-la is conjugated with one or more lipophilic compounds to form a compound of formula la comprising one more lipid conjugates of the disclosure.
  • conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula I-la and one or more fatty acids in series or in parallel by known techniques in the art.
  • conjugation is performed under suitable amide forming conditions to afford a compound of formula I comprising one more lipid conjugates.
  • Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • conjugation of a lipophilic compound can be accomplished by any one of the cross-coupling technologies described in Table A herein.
  • the present disclosure provides a method for preparing an oligonucleotide comprising one or more lipid conjugate, said lipid conjugate unit represent by formula II-l: or a pharmaceutically acceptable salt thereof, comprising the steps of:
  • step (b) conjugating one or more lipophilic compounds to an oligonucleotide of formula II-2 to form an oligonucleotide of formula II-l comprising one or more lipid conjugates.
  • an oligonucleotide of formula II-2 is conjugated with one or more lipophilic compounds to form an oligonucleotide of formula II-l comprising one more lipid conjugates of the disclosure.
  • conjugation is performed through an esterification or amidation reaction between an oligonucleotide of formula II-2 and one or more fatty acids in series or in parallel by known techniques in the art.
  • conjugation is performed under suitable amide forming conditions to afford an oligonucleotide of formula II-l comprising one more lipid conjugates.
  • suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • conjugation of a lipophilic compound can be accomplished by any one of the cross-coupling technologies described in Table A herein.
  • the present disclosure provides a method for preparing an oligonucleotide comprising a unit represent by formula II-2:
  • oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula 1-8 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
  • Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula II-2.
  • the present disclosure provides a method for preparing a nucleic acid or analogue thereof comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula 1-8:
  • PG 1 and PG 2 of a compound of formula 1-1 comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
  • reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A-butyl ammonium fluoride, and the like.
  • a compound of formula 1-6 is protected with a suitable hydroxyl protecting group.
  • the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula 1-6 includes an acid labile protecting group such as trityl, 4-methy oxytrityl, 4,4’ -dimethy oxytrityl, 4,4’,4”-trimethyoxytrityl, 9-phenyl- xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
  • the acid labile protecting group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, dichloroacetic acid or trichloroacetic acid.
  • a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
  • the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
  • the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
  • step (d) above is preformed using 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.
  • the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more adamantyl and/or lipid moieties, said conjugate unit represented by formula II-b-3:
  • oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula Cll is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
  • Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide-ligand conjugate of various nucleotide lengths, with one or more nucleic acid-ligand conjugate units, wherein each unit is represented by a compound of formula II-b-3 comprising an adamantyl or lipid moiety of the disclosure.
  • the method for preparing an oligonucleotide of formula II-b-3 comprising one or more lipid conjugate further comprises preparing a nucleic acid-ligand conjugate or analogue thereof of formula Cll:
  • step (d) treating said nucleic acid-ligand conjugate or analogue thereof of formula C9 with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula Cll.
  • PG 1 and PG 2 of a compound of formula I-b comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
  • reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A- butylammonium fluoride, and the like.
  • a compound of formula C8 is protected with a suitable hydroxyl protecting group.
  • the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula C8 includes an acid labile protecting group such as trityl, 4-methy oxytrityl, 4,4’ -dimethy oxytrityl, 4,4’,4”-trimethyoxytrityl, 9-phenyl- xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
  • the acid labile protecting group is suitable for deprotection during both solutionphase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, di chloroacetic acid or trichloroacetic acid.
  • a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
  • the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
  • the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
  • step (d) above is preformed using A,A-dimethylphosphoramic dichloride as a P(V) forming reagent.
  • the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units each comprising one or more adamantyl or lipid moieties, further comprising preparing a nucleic acid-ligand conjugate or analogue thereof of formula I-b:
  • step (b) conjugating a lipophilic compound to a nucleic acid or analogue thereof of formula C6 to form a nucleic acid-ligand conjugate or analogue thereof of formula I-b comprising one or more adamantyl and/or lipid conjugates.
  • conjugation is performed under suitable amide forming conditions to afford a compound of formula I-b comprising an adamantyl and/or lipid conjugate.
  • Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • the amide forming conditions comprise HATU and DIPEA or TEA.
  • a nucleic acid-ligand conjugate or analogue thereof of formula C6 is provided in salt form (e.g., a fumarate salt) and is first converted to the free base (e.g., using sodium bicarbonate) before preforming the conjugation step.
  • salt form e.g., a fumarate salt
  • free base e.g., sodium bicarbonate
  • the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units, further comprises preparing a nucleic acid-ligand conjugate or analogue thereof of formula C6:
  • step (e) deprotecting said nucleic acid or analogue thereof of formula C5 to form a nucleic acidligand conjugate or analogue thereof of formula C6.
  • step (b) PG 1 and PG 2 groups of formula C2 are taken together with their intervening atoms to form a cyclic diol protecting group, such as a cyclic acetal or ketal.
  • Such groups include methylene, ethylidene, benzylidene, isopropylidene, cyclohexylidene, and cyclopentylidene, silylene derivatives such as di-t-butylsilylene and 1,1,3,3-tetraisopropylidisiloxanylidene, a cyclic carbonate, a cyclic boronate, and cyclic monophosphate derivatives based on cyclic adenosine monophosphate (i.e., cAMP).
  • the cyclic diol protection group is 1, 1,3,3- tetraisopropylidisiloxanylidene prepared from the reaction of a diol of formula Cl and 1,3- dichloro-l,l,3,3-tetraisopropyldisiloxane under basic conditions.
  • a nucleic acid or analogue thereof of formula C2 is alkylated with a mixture of DMSO and acetic anhydride under acidic conditions.
  • a mixture of DMSO and acetic anhydride in the presence of acetic acid forms (methylthio)methyl acetate in situ via the Pummerer rearrangement which then reacts with the hydroxyl group of the nucleic acid or analogue thereof of formula C2 to provide a monothioacetal functionalized fragment nucleic acid or analogue thereof of formula C3.
  • step (d) above substitution of the thiomethyl group of a nucleic acid or analogue thereof of formula C3 using a nucleic acid or analogue thereof of formula C4 affords a nucleic acid or analogue thereof of formula C4.
  • substitution occurs under mild oxidizing and/or acidic conditions.
  • V is oxygen.
  • the mild oxidation reagent includes a mixture of elemental iodine and hydrogen peroxide, urea hydrogen peroxide complex, silver nitrate/silver sulfate, sodium bromate, ammonium peroxodi sulfate, tetrabutylammonium peroxy di sulfate, Oxone®, Chloramine T, Selectfluor®, Selectfluor® II, sodium hypochlorite, or potassium iodate/sodium periodiate.
  • the mild oxidizing agent includes N-iodosuccinimide, N- bromosuccinimide, N-chlorosuccinimide, l,3-diiodo-5,5-dimethylhydantion, pyridinium tribromide, iodine monochloride or complexes thereof, etc.
  • Acids that are typically used under mild oxidizing condition include sulfuric acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid, methanesulfonic acid, and trifluoroacetic acid.
  • the mild oxidation reagent includes a mixture of N-iodosuccinimide and trifluoromethanesulfonic acid.
  • step (e) above removal of PG 3 and optionally R 4 (when R 4 is a suitable amine protecting group) of a nucleic acid-ligand conjugate or analogue thereof of formula C5 affords a nucleic acid-ligand conjugate or analogue thereof of formula C6 or a salt thereof.
  • PG 3 and/or R 4 comprise carbamate derivatives that can be removed under acidic or basic conditions.
  • the protecting groups e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently
  • the protecting groups are removed by acid hydrolysis.
  • a salt of formula C6 thereof is formed upon acid hydrolysis of the protecting groups of a nucleic acid-ligand conjugate or analogue thereof of formula C5, a salt of formula C6 thereof is formed.
  • an acid-labile protecting group of a nucleic acid-ligand conjugate or analogue thereof of formula C5 is removed by treatment with an acid such as hydrochloric acid, then the resulting amine compound would be formed as its hydrochloride salt.
  • acids are useful for removing amino protecting groups that are acid-labile and therefore a wide variety of salt forms of a nucleic acid or analogue thereof of formula C6 are contemplated.
  • the protecting groups e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently
  • the protecting groups are removed by base hydrolysis.
  • Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base.
  • bases are useful for removing amino protecting groups that are base-labile.
  • a base is piperidine.
  • a base is 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU).
  • a nucleic acid-ligand conjugate or analogue thereof of formula C5 is deprotected under basic conditions followed by treating with an acid to form a salt of formula C6.
  • the acid is fumaric acid
  • the salt of formula C6 is the fumarate.
  • the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugate, said nucleic acid-ligand conjugate unit represented by formula II-b-3: or a pharmaceutically acceptable salt thereof, comprising the steps of:
  • step (b) conjugating one or more adamantyl or lipophilic compounds to an oligonucleotide of formula D5 to form an oligonucleotide-ligand conjugate of formula II-b-3 comprising one or more nucleic acid-ligand conjugate units.
  • conjugation is performed under suitable amide forming conditions to afford a compound of formula D5 comprising an adamantyl or lipid conjugate.
  • Suitable amide forming conditions can include the use of an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • an amide coupling reagent known in the art such as, but not limited to HATU, PyBOP, DCC, DIC, EDC, HBTU, HCTU, PyAOP, PyBrOP, BOP, BOP-CI, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • the amide forming conditions comprise HATU and DIPEA or TEA.
  • the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising a unit represent by formula D5: D5 or a salt thereof, comprising the steps of:
  • step (b) deprotecting said compound of formula D4 to form a compound of formula D5.
  • removal of PG 3 and optionally R 4 (when R 4 is a suitable amine protecting group) of an oligonucleotide of formula D4 affords an oligonucleotide-ligand conjugate of formula D5 or a salt thereof.
  • PG 3 and/or R 4 comprise carbamate derivatives that can be removed under acidic or basic conditions.
  • the protecting groups (e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently) of an oligonucleotide- ligand conjugate of formula D4 are removed by acid hydrolysis.
  • the protecting groups e.g., both PG 3 and R 4 or either of PG 3 or R 4 independently
  • the protecting groups are removed by base hydrolysis.
  • Fmoc and trifluoroacetyl protecting groups can be removed by treatment with base.
  • bases are useful for removing amino protecting groups that are base-labile.
  • a base is piperidine.
  • a base is 1,8- diazabicy clo[5.4.0]undec-7-ene (DBU) .
  • the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more nucleic acid-ligand conjugate unit with one or more adamantyl and/or lipid moiety, said conjugate unit represented by formula
  • oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
  • the nucleic acid or analogue thereof of formula D3 is coupled to a solid supported nucleic acid or analogue thereof bearing a 5 ’-hydroxyl group.
  • Further steps can comprise one or more deprotections, couplings, phosphite oxidation, and cleavage from the solid support to provide an oligonucleotide of various nucleotide lengths, represented by a compound of formula D4 comprising an adamantyl or lipid conjugate of the disclosure.
  • the present disclosure provides a method for preparing a nucleic acid or analogue thereof comprising one or more lipid conjugate, further comprising preparing a nucleic acid or analogue thereof of formula D3:
  • PG 1 and PG 2 of a nucleic acid or analogue thereof of formula C5 comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
  • reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-A- butylammonium fluoride, and the like.
  • a nucleic acid or analogue thereof of formula DI is protected with a suitable hydroxyl protecting group.
  • the protecting group PG 4 used for protection of the 5 ’-hydroxyl group of a compound of formula DI includes an acid labile protecting group such as trityl, 4-methy oxytrityl, 4,4’ -dimethy oxytrityl, 4,4’ ,4”- trimethyoxytrityl, 9-phenyl-xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
  • the acid labile protecting group is suitable for deprotection during both solution-phase and solid-phase synthesis of acid-sensitive nucleic acids or analogues thereof using for example, di chloroacetic acid or trichloroacetic acid.
  • a nucleic acid or analogue thereof of formula D2 is treated with a P(III) forming reagent to afford a compound of formula D3.
  • a P(III) forming reagent is a phosphorus reagent that is reacted to for a phosphorus (III) compound.
  • the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
  • the P(IH) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
  • step (d) above is preformed using 7V,7V-dimethylphosphoramic dichloride as a P(V) forming reagent.
  • oligonucleotides e.g., lipid-conjugated RNAi oligonucleotides
  • compositions comprising oligonucleotides reduce the expression of a target mRNA (e.g., a target mRNA expressed in an neurons of the CNS).
  • compositions comprising oligonucleotides (e.g., lipid- conjugated RNAi oligonucleotides) reduce the expression of a target mRNA expressed in one or more tissues or cells of a subject.
  • oligonucleotides e.g., lipid- conjugated RNAi oligonucleotides
  • Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enter the cell to reduce target gene expression.
  • Any variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of target gene expression as disclosed herein.
  • an oligonucleotide is formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.
  • the formulations herein comprise an excipient.
  • an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient.
  • an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).
  • an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject).
  • an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, FicollTM or gelatin).
  • a lyoprotectant e.g., mannitol, lactose, polyethylene glycol or polyvinylpyrrolidone
  • a collapse temperature modifier e.g., dextran, FicollTM or gelatin.
  • the oligonucleotides herein may be provided in the form of their free acids.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intrathecal), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal and rectal administration.
  • a pharmaceutical composition is formulated for delivery to the central nervous system (e.g., intrathecal, epidural).
  • a pharmaceutical composition is formulated for delivery to the eye (e.g., ophthalmic, intraocular, subconjunctival, intravitreal, retrobulbar, intracam eral).
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • a composition may contain at least about 0.1% of the therapeutic agent (e.g., an lipid-conjugated RNAi oligonucleotide herein) or more, although the percentage of the active ingredient(s) may be between about 1% to about 80% or more of the weight or volume of the total composition.
  • the therapeutic agent e.g., an lipid-conjugated RNAi oligonucleotide herein
  • the percentage of the active ingredient(s) may be between about 1% to about 80% or more of the weight or volume of the total composition.
  • Factors such as solubility, bioavailability, biological halflife, route of administration, product shelflife, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • nucleic acids are polymers of subunits or compounds
  • many of the modifications described below occur at a position which is repeated within a nucleic acid (e.g., a modification of a base, or a phosphate moiety, or the non-bridging oxygen of a phosphate moiety).
  • the modification will occur at all of the subject positions in the nucleic acid but in many, and in fact in most cases it will not.
  • a modification may only occur at a 3' or 5' terminal position, may only occur in the internal unpaired region, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand.
  • a modification occurs at all of the subject positions in the nucleic acid.
  • a modification may occur in a double strand region, a single strand region, or in both.
  • a modification may occur only in the double strand region of an RNA agent or may only occur in a single strand region of an RNA agent, e.g., a phosphorothioate modification at a non-bridging oxygen position may only occur at one or both termini, may only occur in a terminal regions or at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini.
  • the 5' end or ends can be phosphorylated.
  • RNAi trigger molecules have not been sufficient for practical therapeutic, research or diagnostic purposes.
  • RNAi trigger molecule oligonucleotides Modifications to enhance the effectiveness of the RNAi trigger molecule oligonucleotides and overcome these problems have taken many forms. These modifications include base ring modifications, sugar moiety modifications, and sugar-phosphate backbone modifications, many exemplified herein and used in the current disclosure. Prior sugarphosphate backbone modifications, particularly on the phosphorus atom, have affected various levels of resistance to nucleases. However, while the ability of an RNAi trigger molecule oligonucleotide to load into the RISC and direct the location of relevant mRNA sequences is fundamental to RNAi trigger molecule methodology, many modifications work at cross purposes with each other to optimize the behavior of the RNAi trigger. It is this balancing act which must be taken into account relative to the development of superior and effective RNAi molecules.
  • phosphorothioate analogs of nucleotides have shown substantial stereoselectivity differences between Oligo-Rp and Oligo- Sp oligonucleotides in resistance to nucleases activity (Potter, BIOCHEMISTRY, 22: 1369, (1983); Bryant et al., BIOCHEMISTRY, 18:2825, (1979)). Lesnikowski (NUCL. ACIDS RES., 18:2109, (1990)) observed that diastereomerically pure octathymidine methylphosphonates, in which six out of seven methylphosphonate bonds have defined configuration at the phosphorus atom when complexed with the matrix showed substantial differences in melting temperatures. According to the current disclosure chirally pure nucleotide analogs, or portions thereof, are expected to provide trigger structures with improved characteristics allowing the development of more potent and longer lasting RNAi triggers.
  • nucleotides or nucleotide surrogates in single strand overhangs, e.g., in a 5' or 3' overhang, or in both.
  • purine nucleotides in overhangs as they are more resistant to nuclease activity.
  • all or some of the bases in a 3' or 5' overhang will be modified, with a modification described herein.
  • Modifications can include the use of modifications at the 2' OH group of the ribose sugar, deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, that is, phosphothioate modifications. Overhangs need not be homologous with the target sequence.
  • the disclosure provides methods for contacting or delivering to a cell or population of cells an effective amount of any of the lipid-conjugated RNAi oligonucleotides herein to reduce expression of a target gene.
  • expression of a target gene is reduced in one or more tissues or cells in a subject.
  • expression of a target gene is reduced in the central nervous system (CNS).
  • CNS central nervous system
  • expression of a target gene is reduced in ocular tissue.
  • expression of a target gene is reduced in the liver.
  • expression of a target gene is reduced in adipose tissue.
  • expression of a target gene is reduced in adrenal tissue.
  • expression of a target gene is reduced in skeletal muscle tissue.
  • expression of a target gene is reduced in the heart.
  • expression of a target gene is reduced in the lung.
  • a reduction of target gene expression is determined by measuring a reduction in the amount or level of target mRNA, protein encoded by the target mRNA, or target gene (mRNA or protein) activity in a cell.
  • the methods include those described herein and known to one of ordinary skill in the art.
  • a cell is any cell that expresses the target mRNA.
  • the cell is a primary cell obtained from a subject.
  • the primary cell has undergone a limited number of passages such that the cell substantially maintains is natural phenotypic properties.
  • a cell to which the oligonucleotide is delivered is ex vivo or in vitro (i.e.. can be delivered to a cell in culture or to an organism in which the cell resides).
  • the lipid-conjugated RNAi oligonucleotides disclosed herein are delivered to a cell or population of cells using a nucleic acid delivery method known in the art including, but not limited to, injection of a solution or pharmaceutical composition containing the lipid-conjugated RNAi oligonucleotide, bombardment by particles covered by the lipid- conjugated RNAi oligonucleotide, exposing the cell or population of cells to a solution containing the lipid-conjugated RNAi oligonucleotide, or electroporation of cell membranes in the presence of the lipid-conjugated RNAi oligonucleotide.
  • Other methods known in the art for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.
  • reduction of target gene expression is determined by an assay or technique that evaluates one or more molecules, properties or characteristics of a cell or population of cells associated with target gene expression, or by an assay or technique that evaluates molecules that are directly indicative of target gene expression in a cell or population of cells (e.g., target mRNA or protein).
  • the extent to which a lipid- conjugated RNAi oligonucleotide provided herein reduces target gene expression in a cell is evaluated by comparing target gene expression in a cell or population of cells contacted with the lipid-conjugated RNAi oligonucleotide to a control cell or population of cells (e.g., a cell or population of cells not contacted with the lipid-conjugated RNAi oligonucleotide or contacted with a control lipid-conjugated RNAi oligonucleotide).
  • a control amount or level of target gene expression in a control cell or population of cells is predetermined, such that the control amount or level need not be measured in every instance the assay or technique is performed.
  • the predetermined level or value can take a variety of forms.
  • a predetermined level or value can be single cut-off value, such as a median or mean.
  • contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene.
  • the reduction in target gene expression is relative to a control amount or level of target gene expression in cell or population of cells not contacted with the lipid-conjugated RNAi oligonucleotide or contacted with a control lipid- conjugated RNAi oligonucleotide.
  • the reduction in target gene expression is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a control amount or level of target gene expression.
  • the control amount or level of target gene expression is an amount or level of target mRNA and/or protein in a cell or population of cells that has not been contacted with a lipid-conjugated RNAi oligonucleotide herein.
  • the effect of delivery of a lipid-conjugated RNAi oligonucleotide to a cell or population of cells according to a method herein is assessed after any finite period or amount of time (e.g., minutes, hours, days, weeks, months).
  • target gene expression is determined in a cell or population of cells at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or more after contacting or delivering the lipid- conjugated RNAi oligonucleotide to the cell or population of cells.
  • target gene expression is determined in a cell or population of cells at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or more after contacting or delivering the lipid-conjugated RNAi oligonucleotide to the cell or population of cells.
  • expression of a target gene is reduced in a region of the CNS.
  • expression of a target gene is reduced in at least one region of the CNS.
  • a region of the CNS is one or more tissues of the CNS.
  • regions of the CNS include, but are not limited to, cerebrum, prefrontal cortex, frontal cortex, motor cortex, temporal cortex, parietal cortex, occipital cortex, somatosensory cortex, hippocampus, caudate, striatum, globus pallidus, thalamus, midbrain, tegmentum, substantia nigra, pons, brainstem, cerebellar white matter, cerebellum, dentate nucleus, medulla, cervical spinal cord, thoracic spinal cord, lumbar spinal cord, cervical dorsal root ganglion, thoracic dorsal root ganglion, lumbar dorsal root ganglion, sacral dorsal root ganglion, nodose ganglia, femoral nerve, sciatic nerve, sural nerve, amygdala, hypothalamus, putamen, corpus callosum, and cranial nerve.
  • the region of the CNS is selected from the spinal cord, lumbar dorsal root ganglion, medulla, hippocampus, frontal cortex, brain stem, cerebellum, and a combination thereof.
  • expression of a target gene is reduced in at least one region of the CNS, selected from frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combination thereof.
  • expression of a neuronal target gene is reduced in at least one tissue of the CNS.
  • expression of an astrocyte target gene is reduced in at least one tissue of the CNS.
  • expression of an oligodendrocyte target gene is reduced in at least one tissue of the CNS.
  • expression of a target mRNA in a neuron is reduced in at least one tissue of the CNS.
  • expression of a target mRNA in an astrocyte is reduced in at least one tissue of the CNS.
  • expression of a target mRNA in an oligodendrocyte is reduced in at least one tissue of the CNS.
  • expression of a target mRNA in a neuron is reduced in at least one tissue of the CNS, selected from frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combination thereof.
  • expression of a target mRNA in an astrocyte is reduced in at least one tissue of the CNS, selected from frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combination thereof.
  • expression of a target mRNA in an oligodendrocyte is reduced in at least one tissue of the CNS, selected from frontal cortex, medulla, hippocampus, hypothalamus, cerebellum, lumbar spinal cord, lumbar dorsal root ganglion, and any combination thereof.
  • expression of a target gene in the CNS of a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to target gene expression in a control tissue.
  • expression of a target gene in an astrocyte of a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to target gene expression in a nontarget cell.
  • expression of a target gene in an oligodendrocyte of a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to target gene expression in a nontarget cell.
  • expression of a target gene in the neurons of a subject is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater than 99% when compared to target gene expression in a nontarget cell.
  • contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene in a neuron.
  • the reduction in expression of a target gene in a neuron is relative to an amount or level of target gene expression in an astrocyte contacted with the lipid-conjugated RNAi oligonucleotide.
  • the reduction expression of a target gene in a neuron is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a level of target gene expression in an astrocyte.
  • the reduction in expression of a target gene in a neuron is relative to an amount or level of target gene expression in an oligodendrocyte contacted with the lipid- conjugated RNAi oligonucleotide.
  • the reduction in expression of a target gene in a neuron is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a level of target gene expression in an oligodendrocyte.
  • reduction in expression of a target gene in an astrocyte is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% relative to reduction in expression of the target gene in a neuron.
  • the reduction in expression of a target gene in an oligodendrocyte is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% relative to reduction in expression of the target gene in a neuron.
  • contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of target gene in an astrocyte.
  • the reduction in expression of a target gene in an astrocyte is relative to an amount or level of target gene expression in a neuron contacted with the lipid-conjugated RNAi oligonucleotide.
  • the reduction in expression of a target gene in an astrocyte is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a level of expression of a target gene in a neuron.
  • the reduction in expression of a target gene in an astrocyte is relative to an amount or level of target gene expression in an oligodendrocytes contacted with the lipid- conjugated RNAi oligonucleotide.
  • the reduction in expression of a target gene in an astrocyte is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a level of target gene expression in an oligodendrocyte.
  • reduction in expression of a target gene in a neuron is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% relative to reduction in expression of the target gene in an astrocyte.
  • the reduction in expression of a target gene in an oligodendrocyte is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% relative to reduction in expression of the target gene in an astrocyte.
  • contacting or delivering a lipid-conjugated RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene in an oligodendrocyte.
  • the reduction in oligodendrocyte target gene expression is relative to an amount or level of target gene expression in neurons contacted with the lipid-conjugated RNAi oligonucleotide.
  • the reduction in a target gene in an oligodendrocyte is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a level of target gene expression in a neuron.
  • the reduction in expression of a target gene in an oligodendrocyte is relative to an amount or level of expression of a target gene in an astrocyte contacted with the lipid-conjugated RNAi oligonucleotide.
  • the reduction in expression of a target gene in an oligodendrocyte is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a level of expression of a target gene in an astrocyte.
  • reduction in expression of a target gene in a neuron is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% relative to reduction in expression of the target gene in an oligodendrocyte.
  • the reduction in expression of a target gene in an astrocyte is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, or at least 90% relative to reduction in expression of the target gene in an oligodendrocyte.
  • contacting or delivering an oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene in the CNS.

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Abstract

L'invention concerne des oligonucléotides conjugués avec des lipides qui inhibent ou réduisent l'expression de gènes cibles. L'invention concerne également des compositions les comprenant et leurs utilisations, en particulier des utilisations se rapportant au traitement de maladies, de troubles et/ou d'états associés à une diminution induite par un déclencheur d'ARNi de l'expression génique cible.
PCT/US2022/049230 2021-11-08 2022-11-08 Conjugués oligonucleotides/arni WO2023081500A2 (fr)

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WO2024040041A1 (fr) * 2022-08-15 2024-02-22 Dicerna Pharmaceuticals, Inc. Régulation de l'activité de molécules d'arni

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KR101147147B1 (ko) * 2004-04-01 2012-05-25 머크 샤프 앤드 돔 코포레이션 Ｒｎａ 간섭의 오프 타겟 효과 감소를 위한 변형된폴리뉴클레오타이드
EP2201022A4 (fr) * 2007-08-27 2012-01-04 Boston Biomedical Inc Composition d'arn duplex asymétrique utile comme mimétique ou inhibiteur de micro arn
CR20210393A (es) * 2018-12-19 2021-10-27 Alnylam Pharmaceuticals Inc COMPOSICIONES DE AGENTE DE ARNi DE PROTEÍNA PRECURSORA DE AMILOIDE (APP) Y MÉTODO DE USO DE LAS MISMAS
AU2020268798A1 (en) * 2019-05-03 2021-11-04 Dicerna Pharmaceuticals, Inc. Double-stranded nucleic acid inhibitor molecules with shortened sense strands

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