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WO2025054459A1 - Conjugués d'oligonucléotides d'arni - Google Patents

Conjugués d'oligonucléotides d'arni Download PDF

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Publication number
WO2025054459A1
WO2025054459A1 PCT/US2024/045600 US2024045600W WO2025054459A1 WO 2025054459 A1 WO2025054459 A1 WO 2025054459A1 US 2024045600 W US2024045600 W US 2024045600W WO 2025054459 A1 WO2025054459 A1 WO 2025054459A1
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Prior art keywords
double
oligonucleotide
nucleotides
stranded oligonucleotide
positions
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PCT/US2024/045600
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English (en)
Inventor
Jose Paredes QUIROZ
Sha DING
Yogesh SHELKE
Xiao-Yi Xiao
Robert V. Kolakowski
Maryam YAHYAEE ANZAHAEE
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Dicerna Pharmaceuticals, Inc.
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Publication of WO2025054459A1 publication Critical patent/WO2025054459A1/fr

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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
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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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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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
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
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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
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present disclosure relates to oligonucleotide-ligand 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. BACKGROUND OF THE DISCLOSURE Regulation of gene expression by modified nucleic acids shows great potential as both a research tool in the laboratory and a therapeutic approach in the clinic.
  • 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 FH12501620.1 Attorney Docket: DCY-13025 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.
  • RNAi duplexes 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.
  • 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) JOURNAL OF HEPATOLOGY 59:1354-59). Technological hurdles remain for the development and clinical use of RNAi oligonucleotides in extrahepatic cells, tissues, and organs. Thus, an ongoing need exists in the art for the successful development of new and effective RNAi oligonucleotides to modulate the expression of a target genes in extrahepatic cells, tissues, and/or 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.
  • RNAi oligonucleotides capable of inhibiting expression of a target gene in extrahepatic tissues while having reduced inhibition in hepatocytes.
  • RNAi oligonucleotides having a ligand comprising a carboxyl conjugated to a nucleotide of the sense strand showed improved efficacy and duration in extrahepatic tissues, including adipose tissue, relative to hepatocytes.
  • target gene expression was reduced in extrahepatic tissue by the RNAi oligonucleotides at a higher amount than reduction of expression of the same target gene in hepatocytes, e.g., reduction by greater than 50% in extrahepatic tissue compared to reduction by 25% in hepatocytes.
  • the disclosure provides a double-stranded oligonucleotide comprising: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-(CO2H)n groups, or a charged form thereof, wherein L is a linker, Y is alkylene, alkenylene, or alkynylene, and n is 1-6, wherein when Y is alkylene, L comprises -O(CH2)a(OCH2CH2)bNH-, wherein a is 1- 6 and b is 0-5, and wherein the ligand is
  • the disclosure provides a double-stranded oligonucleotide comprising: (i) a double-stranded oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length, a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more C6-24 alkylene-(CO2H)n, or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein n is 1-6, wherein knock down of the target mRNA in the extrahepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • the disclosure provides a double-stranded oligonucleotide comprising: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-W n groups, or a charged form thereof, wherein L is a linker, Y is alkylene, alkenylene, or alkynylene, and n is 1-6, wherein when Y is alkylene, L comprises -O(CH2)a(OCH2CH2)bNH- or -O(CH2)a(OCH2CH2)b(NH)(CO)-, wherein a is
  • the disclosure provides a double-stranded oligonucleotide comprising: (i) a double-stranded oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length, a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and FH12501620.1 Attorney Docket: DCY-13025 (ii) one or more ligands, each ligand comprising one or more C 6-24 alkylene-W n , or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein n is 1-6, wherein W is carboxyl, dicarboxyl, -SO 2 OH, tetrazolyl, -OH,
  • n is 1.
  • W is preferably attached to the terminus of the Y group.
  • W is preferably attached to the terminus of an alkylene group.
  • n is 2.
  • one occurrence of W is preferably attached to the terminus of the Y group.
  • one occurrence of W is preferably attached to the terminus of an alkylene group.
  • the other occurrence of W is attached elsewhere in the Y group, most preferably also at the terminus of the Y group, or one atom removed from the terminus of the Y group.
  • each ligand comprises a linker (L), wherein L is conjugated to the one or more C 6-24 alkylene-CO 2 H.
  • L comprises a bivalent or trivalent C1-C50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, - N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, - S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N
  • 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(H)C(O)-, -N(C 1 -C 4 alkyl)C(O)-, -O-, or heteroaryl.
  • the heteroaryl is a triazolyl.
  • the triazolyl is an C 6-24 alkylene, C 6-24 alkenylene, or C 6-24 alkynylene. In some embodiments, Y is an C 6-24 alkylene or C 6-24 alkenylene. In some embodiments, Y is a C 6-24 alkenylene.
  • Y is a C 6-20 alkylene. FH12501620.1 Attorney Docket: DCY-13025
  • the alkenylene comprises from 1-6 olefinic bonds.
  • the alkynylene comprises from 1-6 acetylenic bonds.
  • a is 1.
  • b is 0 or 1.
  • the -L-Y-(CO2H)n group is: , wherein: M is absent, -NRC(O)-, or heteroaryl, wherein R is H or alkyl.
  • c is 0, 1, 2, 3, 4, 5, or 6;
  • d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
  • M is -NRC(O)-.
  • R is H or methyl.
  • R is H.
  • M is heteroaryl.
  • the heteroaryl is triazolyl.
  • the triazolyl is .
  • M is absent.
  • c is 1.
  • d is 0 to 12.
  • d is 4 to 12.
  • d is 4 or 12.
  • d is 0.
  • e is 12 to 23.
  • e is 11, 14, 15, 20, or 21.
  • the extrahepatic tissue is adipose tissue, heart tissue, skeletal muscle, or adrenal gland tissue. In some embodiments, the extrahepatic tissue is adipose tissue.
  • the antisense strand is 22 nucleotides. In some or any of the foregoing or related embodiments, the antisense strand comprises a 3’ overhang of 2 to 6 nucleotides. In some or any of the foregoing or related embodiments, the sense strand and/or antisense strand comprises at least one modified internucleotide linkage. In some embodiments, 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 (i) between positions 1 and 2; (ii) between positions 2 and 3; (iii) between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; or, (iv) between positions 2 and 3, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 36 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, wherein positions are numbered 1-36 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the oligonucleotide comprises a stem-loop comprising a tetraloop.
  • the tetraloop comprises a 5’-GAAA-3’ sequence.
  • the sense strand and/or antisense strand comprises one or more modified nucleotides.
  • the modified nucleotide comprises a modified sugar.
  • the modified sugar comprises a 2’-OMe group.
  • the modified sugar comprises a 2’-F substituent.
  • the antisense strand comprises up to 4 contiguous nucleotides with a 2’-F modified sugar.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, and 14, numbered 5 ⁇ to 3 ⁇ comprise a 2’-F modification.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19, numbered 5 ⁇ to 3 ⁇ , comprise a 2’-F modification.
  • the sense strand comprises 20 nucleotides, wherein nucleotides at each of positions 8, 9, 10, and 11 numbered 5 ⁇ to 3 ⁇ , comprise a 2’-F modification.
  • the sense strand comprises 36 FH12501620.1 Attorney Docket: DCY-13025 nucleotides, wherein nucleotides at each of positions 3, 5, 8, 10, 12, 13, 15, and 17, numbered 5 ⁇ to 3 ⁇ , comprise a 2’-F modification.
  • the antisense strand comprises a 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide. In some or any of the foregoing or related embodiments, the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, and wherein the phosphorylated nucleotide is selected from uridine and adenosine. In some or any of the foregoing or related embodiments, 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 one or more ligands is conjugated to a ribose of the sense strand at the 3’- or 5’-position. In some embodiments, the one or more ligands is conjugated to a ribose of the sense strand at the 2’-position.
  • one ligand is conjugated to position one of the sense strand, numbered 5 ⁇ to 3 ⁇ .
  • the double-stranded oligonucleotide comprises at least two ligands.
  • the at least two ligands are conjugated to different nucleotides of the sense strand.
  • the at least two ligands are the same ligand.
  • the at least two ligands are different ligands.
  • the at least two ligands are conjugated to position 1, numbered 5 ⁇ to 3 ⁇ , and a nucleotide within a tetraloop.
  • At least one ligand is conjugated to position 1 and at least one ligand is conjugated to position 29; (b) at least one ligand is conjugated to position 1 and at least one ligand is conjugated to position 28; (c) at least one ligand is conjugated to position 1 and at least one ligand is conjugated to position 2; or (d) at least one ligand is conjugated to position 1 and at least one ligand is conjugated to position 20, numbered 5 ⁇ to 3 ⁇ , of the sense strand.
  • FH12501620.1 Attorney Docket: DCY-13025
  • the region of complementarity is fully complementary to the mRNA target sequence.
  • the region of complementarity is partially complementary to the mRNA target sequence. In some or any of the foregoing or related embodiments, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (AI): (AI), or a pharmaceutically acceptable salt or charged form thereof, wherein: A and A’ are each independently H or one or more nucleotides; B is a nucleobase; Z is O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl.
  • M is -NRC(O)-.
  • R is H or C1-5 alkyl. In some embodiments, R is H or methyl. In some embodiments, R is H. In some or any of the foregoing or related embodiments, c is 1.
  • the oligonucleotide-ligand conjugate has a structure of Formula (AII) (AII), or a pharmaceutically acceptable salt or charged form thereof.
  • M is heteroaryl.
  • the heteroaryl is triazolyl.
  • the triazolyl is .
  • the oligonucleotide-ligand conjugate has a structure of Formula (AIII) (AIII), or a pharmaceutically acceptable salt or charged form thereof.
  • d is 0 to 12. In some embodiments d is 4 to 12. In some embodiments, d is 4 or 12. In some embodiments, d is 0. In some or any of the foregoing or related embodiments, the oligonucleotide-ligand conjugate has a structure of Formula (AIV) FH12501620.1 Attorney Docket: DCY-13025 (AIV), or a pharmaceutically acceptable salt or a charged form thereof. In some or any of the foregoing or related embodiments, M is absent.
  • the oligonucleotide-ligand conjugate has a structure of Formula (AV) (AV), or a pharmaceutically acceptable salt or a charged form thereof, wherein e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
  • AV Formula
  • a is 1 or 2.
  • a is 1.
  • b is 1 or 2.
  • b is 1.
  • e is 11 to 22. In some embodiments, e is 11, 15, or 21.
  • f is 0, 1, or 2. In some embodiments, f is 0. In some or any of the foregoing or related embodiments, B is FH12501620.1 Attorney Docket: DCY-13025 embodiments, In some or any of the foregoing or related embodiments, A is H. In some or any of the foregoing or related embodiments, Z is S.
  • the disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the oligonucleotide-ligand conjugate of some or any of the foregoing or related embodiments, and wherein the sense strand and antisense strand form a duplex region.
  • the antisense strand is 15 to 30 nucleotides in length.
  • the sense strand and/or antisense strand comprises at least one modified internucleotide linkage.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • A is H and A ⁇ is 12 to 40 nucleotides.
  • the nucleotides of A ⁇ comprise at least one modified internucleotide linkage.
  • A is 12-29 nucleotides and A ⁇ is 1-10 nucleotides.
  • the nucleotides of A comprise at least one modified internucleotide linkage.
  • the oligonucleotide-ligand conjugate and at least 14 nucleotides of A’ form the duplex region with the antisense strand.
  • 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 FH12501620.1 Attorney Docket: DCY-13025 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 (i) between positions 1 and 2; (ii) between positions 2 and 3; (iii) between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; or, (iv) between positions 2 and 3, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 36 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, wherein positions are numbered 1-36 from 5 ⁇ to 3 ⁇ . In some embodiments, the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ . In some or any of the foregoing or related embodiments, the duplex region comprises 20 to 30 base pairs. In some or any of the foregoing or related embodiments, the sense strand comprises a stem-loop comprising a tetraloop.
  • the tetraloop comprises a 5’- GAAA-3’ sequence.
  • the oligonucleotide-ligand conjugate is a nucleotide of the tetraloop.
  • the sense and antisense strand comprise one or more modified nucleotides.
  • the modified nucleotide comprises a modified sugar.
  • the modified sugar comprises a 2’-OMe group.
  • the modified sugar comprises a 2’-F substituent.
  • the sense strand comprises 20 nucleotides, wherein nucleotides at each of positions 8, 9, 10, and 11 comprise a 2’-F modification. In some embodiments, the sense strand comprises 36 nucleotides, wherein nucleotides at each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise a 2’-F modification. In some embodiments, the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2’-F modification.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 comprise a 2’-F modification.
  • the modified nucleotide comprises a modified nucleobase. FH12501620.1 Attorney Docket: DCY-13025
  • the double-stranded oligonucleotide comprises a 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide.
  • the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, and wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • 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 disclosure provides an oligonucleotide-ligand conjugate of Formula (BI) (BI), or a pharmaceutically acceptable salt or charged form thereof, wherein: B is a nucleobase; A is a one or more nucleotides; Z 1 and Z 2 are each independently O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl; a is 1, 2, 3, 4, or 5; c is 0, 1, 2, 3, 4, 5, or 6; d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23; and f is 0, 1, 2, 3, 4, 5, or 6.
  • B is a nucleobase
  • A is a one or more nucleotides
  • Z 1 and Z 2 are each independently O or S
  • M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl
  • a is 1, 2, 3, 4, or
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (BII) (BII), or a salt or charged form thereof, wherein: B is a nucleobase; A is a one or more nucleotides ; Z is O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl; a is 1, 2, 3, 4, or 5; c is 0, 1, 2, 3, 4, 5, or 6; d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 23; and f is 0, 1, 2, 3, 4, 5, or 6.
  • B is a nucleobase
  • A is a one or more nucleotides
  • Z is O or S
  • M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl
  • a is 1, 2, 3, 4, or 5
  • c is 0, 1, 2, 3, 4, 5, or
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (CI) (CI), or a pharmaceutically acceptable salt or charged form thereof, wherein: B is a nucleobase; A is one or more nucleotides; Z 1 and Z 2 are each independently O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl; FH12501620.1 Attorney Docket: DCY-13025 a is 1, 2, 3, 4, or 5; c is 0, 1, 2, 3, 4, 5, or 6; d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 23; and f is 0, 1, 2, 3, 4, 5, or 6.
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (CII) (CII), or a salt or charged form thereof, wherein: B is a nucleobase; A is one or more nucleotides; Z is O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl; a is 1, 2, 3, 4, or 5; c is 0, 1, 2, 3, 4, 5, or 6; d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 23; and f is 0, 1, 2, 3, 4, 5, or 6.
  • CII Formula
  • the disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of some or any of the foregoing or related embodiments, and wherein the sense strand and antisense strand form a duplex region.
  • the antisense strand is 15 to 30 nucleotides in length.
  • A is 12 to 40 nucleotides.
  • the sense strand and/or antisense strand comprises at least one modified internucleotide linkage.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the duplex region includes one or more phosphorothioate linkages.
  • two phosphorothioate linkages are adjacent to each other.
  • 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 (i) between positions 1 and 2; (ii) between positions 2 and 3; (iii) between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; or, (iv) between positions 2 and 3, between positions 18 and 19, and between positiosn 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 36 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, wherein positions are numbered 1-36 from 5 ⁇ to 3 ⁇ . In some embodiments, the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ . In some or any of the foregoing or related embodiments, the duplex region comprises 20 to 30 base pairs. In some or any of the foregoing or related embodiments, the sense strand comprises a loop region that includes a tetraloop region.
  • the tetraloop region comprises a 5’-GAAA-3’ sequence.
  • the sense and antisense strand comprise one or more modified nucleotides.
  • the modified nucleotide comprises a modified sugar.
  • the modified sugar comprises a 2’-OMe group.
  • the modified sugar comprises a 2’-F substituent.
  • the sense strand comprises 20 nucleotides, wherein nucleotides at each of positions 8, 9, 10, and 11 comprise a 2’-F modification.
  • the sense strand comprises 36 nucleotides, wherein nucleotides at each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise a 2’-F modification.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2’-F modification.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 comprise a 2’-F modification.
  • the modified nucleotide comprises a modified nucleobase.
  • the double-stranded oligonucleotide comprises a 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide.
  • the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, and wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • 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 a second oligonucleotide-ligand conjugate.
  • the second oligonucleotide- ligand conjugate is selected from the oligonucleotide-ligand conjugate of some or any of the foregoing or related embodiments.
  • the antisense strand has a region of complementarity to a target sequence in a target mRNA in extrahepatic tissue, wherein the region of complementarity is at least 15 contiguous nucleotides in length. In some embodiments, the region of complementarity is fully complementary to the mRNA target sequence. In some embodiments, the region of complementarity is partially complementary to the mRNA target sequence. In some embodiments, the region of complementarity comprises no more than four mismatches to the mRNA target sequence. In some or any of the foregoing or related embodiments, the extrahepatic tissue is adipose tissue, heart tissue, skeletal muscle, or adrenal gland tissue.
  • the extrahepatic tissue is adipose tissue.
  • the disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises two or more oligonucleotide-ligand conjugates of (i), (ii), or (iii): (i) Formula (AI), (AII), (AIII), (AIV), or (AV); (ii) Formula (BI) or (BII); and FH12501620.1 Attorney Docket: DCY-13025 (iii) Formula (CI) or (CII).
  • the two or more oligonucleotide ligand conjugates are conjugated to different nucleotides of the sense strand. In some embodiments, the two or more oligonucleotide ligand conjugates are the same. In some embodiments, the two or more oligonucleotide ligand conjugates are different. In some or any of the foregoing or related embodiments, the sense strand comprises a first oligonucleotide-ligand conjugate and a second oligonucleotide-ligand conjugate.
  • the nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand and the nucleobase (B) of the second oligonucleotide-ligand conjugate is a nucleobase within a tetraloop of the sense strand.
  • the nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand
  • the nucleobase (B) of the second oligonucleotide-ligand conjugate is the nucleobase at position 29 of the sense strand
  • the nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand
  • the nucleobase (B) of the second oligonucleotide-ligand conjugate is the nucleobase at position 28 of the sense strand
  • the nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand
  • the nucleobase (B) of the second oligonucleotide-ligand conjugate is the nucleobase at position 20 of
  • the oligonucleotide-ligand conjugate reduces expression of the target mRNA in an extrahepatic tissue, provided the oligonucleotide-ligand conjugate does not reduce expression of the mRNA target in the liver.
  • the disclosure provides a pharmaceutical composition comprising the double-stranded oligonucleotide of some or any of the foregoing or related embodiments, and a pharmaceutically acceptable carrier, delivery agent, or excipient.
  • the disclosure provides a method of inhibiting target mRNA expression in an extrahepatic cell or tissue in a subject, comprising administering to the subject the double-stranded oligonucleotide or the pharmaceutical composition of some or FH12501620.1 Attorney Docket: DCY-13025 any of the foregoing or related embodiments, thereby inhibiting target mRNA expression in the cell of the extrahepatic tissue.
  • the extrahepatic cell or tissue is selected from skeletal muscle, adipose tissue, adrenal tissue, heart tissue, and any combination thereof.
  • reduction of the target mRNA in the extrahepatic cell or tissue is increased compared to reduction in liver cells or tissue, optionally wherein reduction of the target mRNA is increased by at least 10%. In some or any of the foregoing or related embodiments, reduction of the target mRNA is increased by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. In some aspects, the disclosure provides the double-stranded oligonucleotide of some or any of the foregoing or related embodiments in the manufacture of a medicament for inhibiting target mRNA expression in an extrahepatic cell or tissue in a subject.
  • the disclosure provides for use of the double-stranded oligonucleotide of some or any of the foregoing or related embodiments for inhibiting target mRNA expression in a cell of an extrahepatic tissue in a subject.
  • the disclosure provides a kit comprising a container comprising the double-stranded oligonucleotide of some or any of the foregoing or related embodiments, and optionally a pharmaceutically acceptable carrier, and instructions for administering the oligonucleotide-ligand conjugate to a subject in need thereof, wherein the oligonucleotide- ligand conjugate inhibits target mRNA expression in an extrahepatic cell or tissue in the subject.
  • the extrahepatic cell or tissue is selected from skeletal muscle, adipose tissue, adrenal tissue, heart tissue, and any combination thereof.
  • the cell of the extrahepatic cell or tissue is selected from a cardiomyocyte, a cell of skeletal muscle, a cell of adipose tissue, a cell of adrenal tissue, and any combination thereof.
  • reduction of the target mRNA in the extrahepatic cell or tissue is increased compared to reduction in a cell of the liver, optionally wherein reduction of the target mRNA is increased by at least 10%.
  • reduction of the target mRNA is increased by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.
  • FH12501620.1 Attorney Docket: DCY-13025
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an mRNA in an extrahepatic cell or tissue, the method comprising administering to the subject a therapeutically effective amount of the double-stranded oligonucleotide or pharmaceutical composition of any or some of the foregoing or related aspects.
  • the disclosure provides a method of delivering a double-stranded oligonucleotide to a cell or population of cells in extrahepatic tissue, the method comprising administering the pharmaceutical composition of any or some of the foregoing or related aspects.
  • the disclosure provides a double-stranded oligonucleotide comprising: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-W n groups, or a charged form thereof, wherein: L is a linker selected from -O(CH 2 ) a (OCH 2 CH 2 ) b NH- , -O(CH2)a(OCH2CH2)b(NH)(CO)-, -O(CH2)a(OCH2CH2)b(NH)(CO)(CH2)c(OCH2CH2)dM-,
  • the disclosure provides a double-stranded oligonucleotide comprising: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-W n groups, or a charged form thereof, wherein: L is a linker selected from -O(CH2)a(OCH2CH2)bNH- , -O(CH 2 ) a (OCH 2 CH 2 ) b (NH)(CO)-, -O(CH 2 ) a (OCH 2 CH 2 ) b (NH)(CO)(CH 2 ) c (OC
  • the disclosure provides double-stranded oligonucleotide comprising: (i) a double-stranded oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length, a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-(C6-24 alkylene)-Wn, or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein: L is a linker selected from -O(CH 2 ) a (OCH 2 CH 2 ) b NH- , -O(CH 2 ) a (OCH 2 CH 2 ) b (
  • L is a linker selected from -O(CH2)a(OCH2CH2)bNH-, -O(CH2)a(OCH2CH2)b(NH)(CO)-, and -O(CH 2 ) a (OCH 2 CH 2 ) b (NH)(CO)(CH 2 ) c (OCH 2 CH 2 ) d M-.
  • L is -O(CH 2 ) a (OCH 2 CH 2 ) b (NH)(CO)(CH 2 ) c (OCH 2 CH 2 ) d M-.
  • L when Y is alkylene, L comprises -O(CH2)a(OCH2CH2)bNH- or -O(CH2)a(OCH2CH2)b(NH)(CO)-.
  • M is heteroarylene.
  • the heteroarylene is a triazolylene.
  • the triazolylene is .
  • M is -NHC(O)-.
  • Y is an C 6-24 alkylene, C 6-24 alkenylene, or C6-24 alkynylene. In some or any of the foregoing or related embodiments, Y is an C6-24 alkylene or C6-24 alkenylene. In some embodiments, Y is a C6-24 alkenylene. In some ebodiments, Y is a C6-20 alkylene. In some embodiments, the alkenylene comprises from 1-6 olefinic bonds. In some embodiments, the alkynylene comprises from 1-6 acetylenic bonds.
  • the alkenylene comprises from 1-6 olefinic bonds; and the alkynylene comprises from 1-6 acetylenic bonds
  • n is 1.
  • W is -CO 2 H, , , , - OH, -N(H)SO2(methyl), -N(H)SO2(ethyl), -N(H)SO2(n-propyl), -N(H)SO2(i-propyl), -N(H)S O2(sec-propyl), -N(H)SO2(n-butyl), -N(H)SO2(i-butyl), -N(H)SO2(sec-butyl), -N(H)SO2(t- butyl), -N(H)SO2(n-pentyl), -N(H)SO2(cyclopropyl), -N
  • a is 1. In some or any of the foregoing or related embodiments, b is 0 or 1. In some or any of the foregoing or related embodiments, b is 2. In some or any of the foregoing or related embodiments, c is 2. In some or any of the foregoing or related embodiments, d is 4. In some or any of the foregoing or related embodiments, the -L-Y-(W) n group is: wherein g is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some or any of the foregoing or related embodiments, M is -NHC(O)-. In some embodiments, M is -N(methyl)C(O).
  • M is heteroarylene.
  • the heteroaryl is triazolylene.
  • the triazolylene is .
  • M is absent.
  • W is -CO2H, , , , - OH, -N(H)SO 2 (methyl), -N(H)SO 2 (ethyl), -N(H)SO 2 (n-propyl), -N(H)SO 2 (i-propyl), -N( H)SO 2 (sec-propyl), -N(H)SO 2 (n-butyl), -N(H)SO 2 (i-butyl), -N(H)SO 2 (sec- butyl), -N(H)SO2(t-butyl), -N(H)SO2(n- pentyl), -N(H)SO2(cyclopropyl), -N(H)SO2(cyclobutyl), -N(H)SO2(cyclopentyl), -N(H)SO2(cyclohexyl
  • c is 1. In some or any of the foregoing or related embodiments, c is 2. In some or any of the foregoing or related embodiments, d is 0 to 12. In some embodiments, d is 4 to 12. In some embodiments, d is 4. In some or any of the foregoing or related embodiments, g is 12 to 23. In some embodiments, g is 11, 14, 15, 20, or 21. FH12501620.1 Attorney Docket: DCY-13025 In some or any of the foregoing or related embodiments, the extrahepatic tissue is adipose tissue, heart tissue, skeletal muscle, or adrenal gland tissue. In some embodiments, the extrahepatic tissue is adipose tissue.
  • the antisense strand is 22 nucleotides. In some or any of the foregoing or related embodiments, the antisense strand comprises a 3’ overhang of 2 to 6 nucleotides. In some or any of the foregoing or related embodiments, the sense strand and/or antisense strand comprises at least one modified internucleotide linkage. In some embodiments, 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’.
  • 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 (i) between positions 1 and 2; (ii) between positions 2 and 3; (iii) between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; or, (iv) between positions 2 and 3, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 36 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, wherein positions are numbered 1-36 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the oligonucleotide comprises a stem-loop comprising a tetraloop.
  • the tetraloop comprises a 5’-GAAA-3’ sequence.
  • the sense strand and/or antisense strand comprises one or more modified nucleotides.
  • the modified nucleotide comprises a modified sugar.
  • the modified sugar comprises a 2’-OMe group. In some embodiments, the modified sugar comprises a 2’-F substituent. FH12501620.1 Attorney Docket: DCY-13025
  • the antisense strand comprises up to 4 contiguous nucleotides with a 2’-F modified sugar. In some embodiments, the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, and 14, numbered 5 ⁇ to 3 ⁇ comprise a 2’-F modification.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19, numbered 5 ⁇ to 3 ⁇ , comprise a 2’-F modification.
  • the sense strand comprises 20 nucleotides, wherein nucleotides at each of positions 8, 9, 10, and 11 numbered 5 ⁇ to 3 ⁇ , comprise a 2’-F modification.
  • the sense strand comprises 36 nucleotides, wherein nucleotides at each of positions 3, 5, 8, 10, 12, 13, 15, and 17, numbered 5 ⁇ to 3 ⁇ , comprise a 2’-F modification.
  • the antisense strand comprises a 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide. In some or any of the foregoing or related embodiments, the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, and wherein the phosphorylated nucleotide is selected from uridine and adenosine. In some or any of the foregoing or related embodiments, the 4 ⁇ -carbon of the sugar of the 5 ⁇ -nucleotide of the antisense strand comprises a phosphate analog.
  • phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate.
  • the phosphorylated nucleotide is 4’-O-monomethylphosphonate-2’-O-methyl uridine.
  • the one or more ligands is conjugated to a ribose of the sense strand at the 3’- or 5’-position. In some or any of the foregoing or related embodiments, the one or more ligands is conjugated to a ribose of the sense strand at the 2’-position.
  • one ligand is conjugated to position one of the sense strand, numbered 5 ⁇ to 3 ⁇ .
  • the double-stranded oligonucleotide comprises at least two ligands.
  • the at least two ligands are conjugated to different nucleotides of the sense strand.
  • the at least two ligands are the same ligand.
  • the at least two ligands are different ligands.
  • the at least two ligands are conjugated to position 1, numbered 5 ⁇ to 3 ⁇ , and a nucleotide within a tetraloop.
  • the region of complementarity is partially complementary to the mRNA target sequence. In some or any of the foregoing or related embodiments, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (AI): or a pharmaceutically acceptable salt or charged form thereof; wherein: A and A’ are each independently H or one or more nucleotides; B is a nucleobase; Z is O or S; M is absent, -NRC(O)-, or heteroarylene, wherein R is H or alkyl; each W is independently -C(O)OH, -CR ⁇ (C(O)OH) 2 , -CR ⁇ (C(O)O(alkyl)) 2 , -SO 2 OH, tetrazolyl, -OH, -N(H)SO 2 (alkyl), -N(H)SO 2 (cycloalkyl), -N(H)SO 2 (optionally substituted aryl), FH12501620.1 Attorney Docket: DCY-13025 -N(H)SO2(heterocyclyl), -N(H)SO2(optionally
  • M is -NRC(O)-.
  • R is H or C1-5 alkyl. In some embodiments, R is H or methyl. In some embodiments, R is H.
  • W is -CO 2 H, , , , - OH, -N(H)SO2(methyl), -N(H)SO2(ethyl), -N(H)SO2(n-propyl), -N(H)SO2(i-propyl), -N( H)SO2(sec-propyl), -N(H)SO2(n-butyl), -N(H)SO2(i-butyl), -N(H)SO2(sec- butyl), -N(H)SO2(t-butyl), -N(H)SO2(n- pentyl), -N(H)SO2(cyclopropyl), -N(H)SO2(cyclobutyl), -N(H)SO2(cyclopentyl), -N(H)SO2(cyclohexyl), -N(H)SO2(cycloheptyl
  • c is 1 or 2.
  • the oligonucleotide-ligand conjugate has a structure of Formula (AII) or a pharmaceutically acceptable salt or charged form thereof.
  • M is heteroarylene.
  • the heteroarylene is triazolylene. In some embodiments, the triazolylene is .
  • the oligonucleotide-ligand conjugate has a structure of Formula (AIII) FH12501620.1 Attorney Docket: DCY-13025 or a pharmaceutically acceptable salt or charged form thereof.
  • d is 0 to 12. In some embodiments, d is 4 to 12. In some embodiments, d is 4. In some or any of the foregoing or related embodiments, the oligonucleotide-ligand has a structure of Formula (AIV) or a pharmaceutically acceptable salt or a charged form thereof. In some or any of the foregoing or related embodiments, M is absent.
  • the oligonucleotide-ligand conjugate has a structure of Formula (AV) or a pharmaceutically acceptable salt or a charged form thereof.
  • AV Formula
  • a is 1 or 2.
  • a is 1.
  • FH12501620.1 Attorney Docket: DCY-13025
  • b is 1 or 2.
  • b is 2.
  • e is 11 to 22.
  • e is 11, 15, or 21.
  • f is 0, 1, or 2.
  • f is 0. .
  • A is H.
  • Z is S.
  • the disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises the oligonucleotide-ligand conjugate of some or any of the foregoing or related embodiments, and wherein the sense strand and antisense strand form a duplex region.
  • the antisense strand is 15 to 30 nucleotides in length.
  • the sense strand and/or antisense strand comprises at least one modified internucleotide linkage.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • A is H and A ⁇ is 12 to 40 nucleotides.
  • the nucleotides of A ⁇ comprise at least one modified internucleotide linkage.
  • A is 12-29 nucleotides and A ⁇ is 1-10 nucleotides.
  • the nucleotides of A comprise at least one modified internucleotide linkage.
  • the oligonucleotide- ligand conjugate and at least 14 nucleotides of A’ form the duplex region with the antisense strand.
  • 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 (i) between positions 1 and 2; (ii) between positions 2 and 3; (iii) between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; or, (iv) between positions 2 and 3, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 36 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, wherein positions are numbered 1-36 from 5 ⁇ to 3 ⁇ . In some or any of the foregoing or related embodiments, the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ . In some or any of the foregoing or related embodiments, the duplex region comprises 20 to 30 base pairs.
  • the sense strand comprises a stem-loop comprising a tetraloop.
  • the tetraloop comprises a 5’- GAAA-3’ sequence.
  • the oligonucleotide-ligand conjugate is a nucleotide of the tetraloop.
  • the sense and antisense strand comprise one or more modified nucleotides.
  • the modified FH12501620.1 Attorney Docket: DCY-13025 nucleotide comprises a modified sugar.
  • the modified sugar comprises a 2’-OMe group.
  • the modified sugar comprises a 2’-F substituent.
  • the sense strand comprises 20 nucleotides, wherein nucleotides at each of positions 8, 9, 10, and 11 comprise a 2’-F modification.
  • the sense strand comprises 36 nucleotides, wherein nucleotides at each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise a 2’-F modification.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2’-F modification.
  • antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 comprise a 2’-F modification.
  • the modified nucleotide comprises a modified nucleobase.
  • the double-stranded oligonucleotide comprises a 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide.
  • the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, and wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • 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 disclosure provides an oligonucleotide-ligand conjugate of Formula (BI): FH12501620.1 Attorney Docket: DCY-13025 or a pharmaceutically acceptable salt or charged form thereof, wherein: B is a nucleobase; A is a one or more nucleotides; Z 1 and Z 2 are each independently O or S; M is absent, -NRC(O)-, or heteroarylene, wherein R is H or alkyl; W is independently -C(O)OH, -CR ⁇ (C(O)OH) 2 , -CR ⁇ (C(O)O(alkyl)) 2 , -SO 2 OH, tetrazolyl, -OH, -N(H)SO 2 (alkyl), -N(H)SO 2 (cycloalky
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (BII): FH12501620.1 Attorney Docket: DCY-13025 or a salt or charged form thereof, wherein: B is a nucleobase; A is a one or more nucleotides ; Z is O or S; M is absent, NRC(O), or heteroarylene, wherein R is H or alkyl; W is independently -C(O)OH, -CR ⁇ (C(O)OH)2, -CR ⁇ (C(O)O(alkyl))2, -SO2OH, tetrazolyl, -OH, -N(H)SO2(alkyl), -N(H)SO2(cycloalkyl), -N(H)SO2(optionally substituted aryl), -N(H)SO 2 (heterocyclyl), -N(H)SO 2 (optionally substituted heteroaryl), -O(optionally substituted aryl),
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (CI) or a pharmaceutically acceptable salt or charged form thereof, wherein: FH12501620.1 Attorney Docket: DCY-13025 B is a nucleobase; A is one or more nucleotides; Z 1 and Z 2 are each independently O or S; M is absent, NRC(O), or heteroarylene, wherein R is H or alkyl; W is independently -C(O)OH, -CR ⁇ (C(O)OH)2, -CR ⁇ (C(O)O(alkyl))2, -SO2OH, tetrazolyl, -OH, -N(H)SO2(alkyl), -N(H)SO2(cycloalkyl), -N(H)SO2(optionally substituted aryl), -N(H)SO2(heterocyclyl), -N(H)SO2(optionally substituted heteroaryl), -O(optionally
  • the disclosure provides an oligonucleotide-ligand conjugate of Formula (CII) or a salt or charged form thereof, wherein: B is a nucleobase; A is one or more nucleotides; Z is O or S; M is absent, NRC(O), or heteroarylene, wherein R is H or alkyl; W is independently -C(O)OH, -CR ⁇ (C(O)OH) 2 , -CR ⁇ (C(O)O(alkyl)) 2 , -SO 2 OH, tetrazolyl, -OH, -N(H)SO 2 (alkyl), -N(H)SO 2 (cycloalkyl), -N(H)SO 2 (optionally substituted aryl), -N(H)SO2(heterocyclyl), -N(H)SO2(optionally substituted heteroaryl), -O(optionally substituted FH12501620.1 Attorney Docket: DCY-13025
  • the disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of some or any of the foregoing or related embodiments, and wherein the sense strand and antisense strand form a duplex region.
  • the antisense strand is 15 to 30 nucleotides in length.
  • A is 12 to 40 nucleotides.
  • the sense strand and/or antisense strand comprises at least one modified internucleotide linkage.
  • the at least one modified internucleotide linkage is a phosphorothioate linkage.
  • the duplex region includes one or more phosphorothioate linkages.
  • two phosphorothioate linkages are adjacent to each other.
  • 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 (i) between positions 1 and 2; (ii) between positions 2 and 3; (iii) between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20; or, (iv) between positions 2 and 3, between positions 18 and 19, and between positiosn 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 36 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, wherein positions are numbered 1-36 from 5 ⁇ to 3 ⁇ .
  • the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • the duplex region comprises 20 to 30 base pairs.
  • the sense strand comprises a loop region that includes a tetraloop region. In some embodiments, the tetraloop region comprises a 5’-GAAA-3’ sequence. In some or any of the foregoing or related embodiments, the sense and antisense strand comprise one or more modified nucleotides. In some embodiments, the modified nucleotide comprises a modified sugar. In some embodiments, the modified sugar comprises a 2’-OMe group. In some embodiments, the modified sugar comprises a 2’-F substituent.
  • the sense strand comprises 20 nucleotides, wherein nucleotides at each of positions 8, 9, 10, and 11 comprise a 2’-F modification. In some or any of the foregoing or related embodiments, the sense strand comprises 36 nucleotides, wherein nucleotides at each of positions 3, 5, 8, 10, 12, 13, 15, and 17 comprise a 2’-F modification. In some or any of the foregoing or related embodiments, the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2’-F modification.
  • the antisense strand comprises 22 nucleotides, wherein nucleotides at each of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 comprise a 2’-F modification.
  • the modified nucleotide comprises a modified nucleobase.
  • the double-stranded oligonucleotide of comprises a 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide.
  • the antisense strand comprises a phosphorylated nucleotide at the 5’ terminus, and wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • 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 a second oligonucleotide-ligand conjugate.
  • the second oligonucleotide- ligand conjugate is selected from the oligonucleotide-ligand conjugate of any some or any of the foregoing or related aspects.
  • the antisense strand has a region of complementarity to a target sequence in a target mRNA in extrahepatic tissue, wherein the region of complementarity is at least 15 contiguous nucleotides in length. In some or any of the foregoing or related embodiments, the region of complementarity is fully complementary to the mRNA target sequence. In some or any of the foregoing or related embodiments, the region of complementarity is partially complementary to the mRNA target sequence. In some or any of the foregoing or related embodiments, the region of complementarity comprises no more than four mismatches to the mRNA target sequence.
  • the extrahepatic tissue is adipose tissue, heart tissue, skeletal muscle, or adrenal gland tissue. In some embodiments, the extrahepatic tissue is adipose tissue.
  • the disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, wherein the sense strand comprises two or more oligonucleotide-ligand conjugates of (i), (ii), or (iii): (i) Formula (AI), (AII), (AIII), (AIV), or (AV); (ii) Formula (BI) or (BII); and (iii) Formula (CI) or (CII).
  • the two or more oligonucleotide ligand conjugates are conjugated to different nucleotides of the sense strand.
  • FH12501620.1 Attorney Docket: DCY-13025
  • the two or more oligonucleotide ligand conjugates are the same.
  • the two or more oligonucleotide ligand conjugates are different.
  • the sense strand comprises a first oligonucleotide-ligand conjugate and a second oligonucleotide-ligand conjugate.
  • nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand and the nucleobase (B) of the second oligonucleotide-ligand conjugate is a nucleobase within a tetraloop of the sense strand.
  • the nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand
  • the nucleobase (B) of the second oligonucleotide-ligand conjugate is the nucleobase at position 29 of the sense strand
  • the nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand
  • the nucleobase (B) of the second oligonucleotide-ligand conjugate is the nucleobase at position 28 of the sense strand
  • the nucleobase (B) of the first oligonucleotide-ligand conjugate is the nucleobase at position 1 of the sense strand
  • the nucleobase (B) of the second oligonucleotide-ligand conjugate is the nucleobase at position 20 of
  • the oligonucleotide-ligand conjugate reduces expression of the target mRNA in an extrahepatic tissue, provided the oligonucleotide-ligand conjugate does not reduce expression of the mRNA target in the liver.
  • the disclosure provides a pharmaceutical composition comprising the double-stranded oligonucleotide of some or any of the foregoing or related embodiments, and a pharmaceutically acceptable carrier, delivery agent, or excipient.
  • the disclosure provides a method of inhibiting target mRNA expression in an extrahepatic cell or tissue in a subject, comprising administering to the subject the double-stranded oligonucleotide of some or any of the foregoing or related embodiments, or the pharmaceutical composition of any of the foregoing or related FH12501620.1 Attorney Docket: DCY-13025 embodiments, thereby inhibiting target mRNA expression in the cell of the extrahepatic tissue.
  • the extrahepatic cell or tissue is selected from skeletal muscle, adipose tissue, adrenal tissue, heart tissue, and any combination thereof.
  • reduction of the target mRNA in the extrahepatic cell or tissue is increased compared to reduction in liver cells or tissue, optionally wherein reduction of the target mRNA is increased by at least 10%. In some or any of the foregoing or related embodiments, reduction of the target mRNA is increased by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. In some aspects, the disclosure provides the double-stranded oligonucleotide of any one of the foregoing or related embodiments in the manufacture of a medicament for inhibiting target mRNA expression in an extrahepatic cell or tissue in a subject.
  • the disclosure provides for use of the double-stranded oligonucleotide of any one of the foregoing or related embodiments for inhibiting target mRNA expression in a cell of an extrahepatic tissue in a subject.
  • the disclosure provides a kit comprising a container comprising the double-stranded oligonucleotide of some or any of the foregoing or related embodiments, and optionally a pharmaceutically acceptable carrier, and instructions for administering the oligonucleotide-ligand conjugate to a subject in need thereof, wherein the oligonucleotide- ligand conjugate inhibits target mRNA expression in an extrahepatic cell or tissue in the subject.
  • the extrahepatic cell or tissue is selected from skeletal muscle, adipose tissue, adrenal tissue, heart tissue, and any combination thereof.
  • the cell of the extrahepatic cell or tissue is selected from a cardiomyocyte, a cell of skeletal muscle, a cell of adipose tissue, a cell of adrenal tissue, and any combination thereof.
  • reduction of the target mRNA in the extrahepatic cell or tissue is increased compared to reduction in a cell of the liver, optionally wherein reduction of the target mRNA is increased by at least 10%.
  • reduction of the target mRNA is increased by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.
  • the disclosure provides a method for treating a subject having a disease, disorder or condition associated with expression of an mRNA in an extrahepatic cell or tissue, the method comprising administering to the subject a therapeutically effective amount of the double-stranded oligonucleotide of some or any of the foregoing or related embodiments, or the pharmaceutical composition of any of the foregoing or related embodiments.
  • the disclosure provides a method of delivering a double-stranded oligonucleotide to a cell or population of cells in extrahepatic tissue, the method comprising administering the pharmaceutical composition of some or any of the foregoing or related embodiments.
  • FIG.1A provides structures of alkyl and alkylene-COOH groups suitable for conjugation to RNAi oligonucleotides.
  • FIG.1B provides the structure of RNAi oligonucleotides having chemical modifications with a C22 conjugate linked to a nucleotide in a stem loop (Duplex A); a C16- COOH conjugate linked to a nucleotide in a stem loop (Duplex B); a C16-COOH conjugate linked to position 1 (P1) of the sense strand (Duplex C); or, a C16 conjugate linked to P1 of the sense strand and a C22 conjugate linked to a nucleotide in a the stem loop (Duplex D).
  • FIGs.2A-2B are graphs demonstrating remaining mouse Aldh2 mRNA levels in the liver (FIG.2A) or adipose tissue (FIG.2B) of mice following intravenous treatment with a single dose (15mg/kg) of the corresponding oligonucleotide in FIG.1B. Tissue was collected 7 days post administration. Control mice were administered PBS.
  • FIG.3 provides the structure of RNAi oligonucleotide molecules comprising a C22 conjugate (Duplex A) or a C16-COOH conjugate (Duplex B) linked to the stem loop of the sense strand.
  • FIGs.4A-4D are graphs demonstrating remaining mouse Aldh2 mRNA levels in the liver (FIG.4A), gonadal white adipose tissue (gWAT) (FIG.4B), quadricep (FIG.4C), and subcutaneous white adipose tissue (scWAT) (FIG.4D) of mice following intravenous treatment with a single dose (15 mg/kg) of a corresponding oligonucleotide in FIG.3. Tissue was collected 7 days post administration. Control mice were administered PBS. FH12501620.1 Attorney Docket: DCY-13025 FIG.5 provides structures of RNAi oligonucleotide conjugates.
  • FIGs.6A-6F are graphs demonstrating remaining mouse Aldh2 mRNA levels in the liver (FIG.6A), scWAT (FIG.6B), gWAT (FIG.6C), heart (FIG.6D), quadricep (FIG. 6E), and adrenal tissue (FIG.6F) of mice following intravenous treatment with a single dose (15 mg/kg) of oligonucleotides comprising a modified sense strand of SEQ ID NO: 2 or 4 and a modified antisense strand of SEQ ID NO: 9 linked to conjugates as depicted in FIG.5 and the parent C22 conjugate shown in FIG.1B.
  • FIG.7 provides structures of RNAi oligonucleotide conjugates. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.8A-8D are graphs demonstrating remaining mouse Aldh2 mRNA levels in the liver (FIG.8A), adipose tissue (FIG.8B), quadricep (FIG.8C), and heart (FIG.8D) of mice following subcutaneous treatment with a single dose (10 mg/kg) of oligonucleotides comprising a modified sense strand of SEQ ID NO: 10 and a modified antisense strand of SEQ ID NO: 5 or 6 linked to conjugates as depicted in FIG.7 and the parent C22 conjugate shown in FIG.1B.
  • FIG.9 provides structures of RNAi oligonucleotide conjugates. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.10A-10D are graphs demonstrating remaining mouse Aldh2 mRNA levels in the liver (FIG.10A), skeletal muscle (FIG.10B), heart (FIG.10C), and gWAT (FIG.10D) of mice following subcutaneous treatment with a single dose (10 mg/kg) of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.9.
  • FIGs.11A-11D are graphs demonstrating remaining mouse Aldh2 mRNA levels in the liver (FIG.11A), skeletal muscle (FIG.11B), heart (FIG.11C), and gWAT (FIG.11D) of mice following subcutaneous treatment with a single dose (10 mg/kg) of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.9. Tissue was collected 21 days post administration. Control mice were administered PBS.
  • FIG.12 provides structures of RNAi oligonucleotide molecules comprising a C16 conjugate and/or a C16-COOH conjugate at different positions of the sense strand. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.13A-13D are graphs demonstrating remaining mouse Aldh2 mRNA levels in the liver (FIG.13A), quadricep (FIG.13B), heart (FIG.13C), and adipose tissue (FIG.
  • FIG.14 provides structures of RNAi oligonucleotide conjugates. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.15A-15B are graphs demonstrating remaining monkey Aldh2 mRNA levels in liver (FIG.15A) and muscle (FIG.15B) of cynomolgus monkeys following subcutaneous treatment with a single dose 10 mg/kg of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.14. Tissue was collected 28 days post administration. Control animals were administered PBS.
  • FIG.16 provides structures of RNAi oligonucleotide conjugates. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.17A-17D are graphs demonstrating remaining monkey Aldh2 mRNA levels in liver (FIG.17A), skeletal muscle (FIG.17B), heart (FIG.17C), and gWAT (FIG.17D) of cynomolgus monkeys following subcutaneous treatment with a single dose 10 mg/kg of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.16. Tissue was collected 28 days post administration. Control animals were administered PBS.
  • FIG.18 provides structures of RNAi oligonucleotide conjugates.
  • FIGs.19A-19D are graphs demonstrating remaining monkey Aldh2 mRNA levels in liver (FIG.19A), skeletal muscle (FIG.19B), heart (FIG.19C), and gWAT (FIG.19D) of cynomolgus monkeys following subcutaneous treatment with a single dose 10 mg/kg of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.18. Tissue was collected 28 days post administration. Control animals were administered PBS.
  • FIG.20 provides structures of RNAi oligonucleotide conjugates. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.21A-21D are graphs demonstrating remaining monkey Aldh2 mRNA levels in liver (FIG.21A), skeletal muscle (FIG.21B), heart (FIG.21C), and gWAT (FIG.21D) of cynomolgus monkeys following subcutaneous treatment with a single dose 10 mg/kg of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.20.
  • FIG.22 provides structures of RNAi oligonucleotide conjugates. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.23A-23D are graphs demonstrating remaining monkey Aldh2 mRNA levels in liver (FIG.23A), skeletal muscle (FIG.23B), heart (FIG.23C), and gWAT (FIG.23D) of cynomolgus monkeys following subcutaneous treatment with a single dose 10 mg/kg of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.22.
  • FIG.24 provides structures of RNAi oligonucleotide conjugates. The key for the depicted molecules is the same as that provided in FIG.1B.
  • FIGs.25A-25D are graphs demonstrating remaining monkey Aldh2 mRNA levels in liver (FIG.25A), skeletal muscle (FIG.25B), heart (FIG.25C), and gWAT (FIG.25D) of cynomolgus monkeys following subcutaneous treatment with a single dose 10 mg/kg of oligonucleotides comprising a modified sense strand of SEQ ID NO: 5 or 7 and a modified antisense strand of SEQ ID NO: 10 linked to conjugates as depicted in FIG.24.
  • the disclosure provides oligonucleotide-conjugates (e.g., RNAi oligonucleotides comprising an oligonucleotide-ligand conjugate) that reduce expression of a target gene in extrahepatic tissue.
  • oligonucleotide-conjugates e.g., RNAi oligonucleotides comprising an oligonucleotide-ligand conjugate
  • 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 associated with expression of a target gene using the oligonucleotide-conjugates, or pharmaceutically acceptable compositions thereof, described herein.
  • the disclosure provides methods of using the FH12501620.1 Attorney Docket: DCY-13025 oligonucleotide-conjugates described herein in the manufacture of a medicament for treating a disease or disorder associated with expression of a target gene.
  • the oligonucleotide-conjugates described herein comprise one or more ligands bearing carboxyl groups.
  • the ligands comprise alkylene, alkenylene, or alkynylene groups, and one or more carboxyl groups are located on the alkylene, alkenylene, or alkynylene (referred to interchangeably herein as a “hydrocarbon chain bearing one or more carboxyl groups’).
  • such ligands containing hydrocarbon chains bearing one or more carboxyl groups target extrahepatic tissue, and thus are capable of modulating the expression of a target genes in extrahepatic cells, tissues, and/or organs.
  • the size of the alkylene, alkenylene or alkynylene chain bearing the carboxyl group was discovered to be important in targeting extrahepatic tissue. More specifically, when carboxylic acids were located on hydrocarbon chains having from 6 to 24 carbons (not including the number of carbons in the linker as described here), the conjugates effectively modified expression of a target gene in extrahepatic tissue at a higher amount than reduction of expression of the same target gene in hepatocytes.
  • oligonucleotide-conjugates to be used in treating disease with specificity for extrahepatic tissue.
  • nucleic acid chemistry many different artificial nucleic acids have been developed to alter the behavior of siRNAs under physiological conditions.
  • phosphorothioate (PS), 2 ⁇ -methoxy (2 ⁇ -OMe), and 2 ⁇ -fluoro nucleic acid have often been used to modify the siRNA its behavior, toxicity and thermostability.
  • Oligonucleotide-Ligand Conjugates The disclosure provides, inter alia, RNAi oligonucleotides comprising at least one oligonucleotide-ligand conjugate that reduce expression of a target gene in extrahepatic tissue.
  • an RNAi oligonucleotide provided by the disclosure targets an mRNA encoding the target gene.
  • Messenger RNA (mRNA) that encodes a target gene and is targeted by an RNAi oligonucleotide of the disclosure is referred to herein as “target mRNA”.
  • the ligand comprises one or more Y-(CO 2 H) n groups, or a charged form thereof, wherein Y is alkylene, alkenylene, or alkynylene, and n is 1-6.
  • the alkylene, alkenylene, or alkynylene comprises at least 5, at least 6, at least 7 at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26 carbon atoms.
  • the alkylene, alkenylene, or FH12501620.1 Attorney Docket: DCY-13025 alkynylene comprises from 6-26 carbon atoms (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, including all values and ranges therein), or from 10-22 carbon atoms (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, including all values and ranges therein).
  • the alkylene, alkenylene, or alkynylene comprises at least 6 (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26) carbon atoms.
  • the alkylene, alkenylene, or alkynylene comprises less than or equal to 30, less than or equal to 29, less than or equal to 28, less than or equal to 27, less than or equal to 26, or less than equal to 25 carbon atoms.
  • n is 1, 2, 3, 4, 5 or 6. In some embodiments, n is 1 or 2.
  • the ligand comprises 1, 2, 3, 4, 5, 6 or more Y-(CO2H)n groups, or a charged form thereof.
  • the oligonucleotide comprises 1, 2, 3, 4, 5, or more ligands.
  • the ligand further comprises a linker moiety, L.
  • L comprises a bivalent or trivalent C 1 -C 50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C 1 -C 4 alkyl)-, -N(cycloalkyl)- , -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, - N(H)C(O)-, -N(C 1 -C 4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C 1 -C 4 alkyl), - C(O)N(cycloalkyl), aryl, heteroaryl
  • b is 0, 1, 2, 3, 4 or 5.
  • L may further comprises a total of 50 carbon atoms in which up to 25 methylene groups are optionally and independently replaced as described above.
  • L when Y is alkylene, L comprises -O(CH2)a(OCH2CH2)bNH-, wherein a is 1-6 and b is 0-5, and optionally the methylene groups and substitutions mentioned above, such that L comprises up to C1-C50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced as described above.
  • a is 1, 2, 3, 4, 5 or 6.
  • a is 2.
  • b is 1, 2, 3, 4, 5 or 6.
  • an RNAi oligonucleotide comprising: (i) an antisense strand of 15 to 30 nucleotides in length and a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-(CO2H)n groups, or a charged form thereof, wherein L is a linker, Y is alkylene, alkenylene, or alkynylene, and n FH12501620.1 Attorney Docket: DCY-13025 is 1-6, wherein when Y is alkylene, L comprises -O(CH 2 ) a (OC
  • the RNAi oligonucleotide comprises: (i) an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more C6-24 alkylene-(CO2H)n, or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein n is 1-6, wherein knock down of the target mRNA in the extrahepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • the extra-hepatic tissue is adipose, heart, skeletal muscle, the central nervous system, or adrenal gland tissue. In some embodiments, the extrahepatic tissue is adipose tissue. In some embodiments, the extrahepatic tissue is heart tissue. In some embodiments, the extrahepatic tissue is skeletal muscle. In some embodiments, the extrahepatic tissue is adrenal gland tissue. In some embodiments, the extrahepatic tissue is central nervous system tissue.
  • the RNAi oligonucleotide comprises an antisense strand of about 15 to 30 nucleotides in length, for example, about 15 to 30 nucleotides in length, about 15 to 29 nucleotides in length, about 15 to 28 nucleotides in length, about 15 to 27 nucleotides in length, about 15 to 26 nucleotides in length, about 15 to 25 nucleotides in length, about 15 to 24 nucleotides in length, about 15 to 23 nucleotides in length, about 15 to 22 nucleotides in length, about 15 to 21 nucleotides in length, about 15 to 20 nucleotides in length, including any values or ranges therebetween.
  • the antisense strand comprises 22 nucleotides.
  • the RNAi oligonucleotide comprises a sense strand of about 13 to 40 nucleotides in length, for example, about 13 to 40 nucleotides in length, about 13 to 38 nucleotides in length, about 13 to 36 nucleotides in length, about 13 to 34 nucleotides in length, about 13 to 32 nucleotides in length, about 13 to 30 nucleotides in length, about 13 to 28 nucleotides in length, about 15 to 40 nucleotides in length, about 15 to 38 nucleotides in length, about 15 to 36 nucleotides in length, about 15 to 34 nucleotides in length, about 15 to 32 nucleotides in length, about 15 to 30 nucleotides in length, about 15 to 28 nucleotides in length, about 20 to 40 nucleotides in length, about 20 to 38 nucleotides in length, about 20 to 36 nucleotides in length,
  • the sense strand comprises 28 nucleotides.
  • each of the one or more ligands comprises one or more -L-Y- CO 2 H group of a charged form thereof.
  • each of the one or more ligands comprises two or more -L-Y-CO2H group of a charged form thereof.
  • each of the three or more ligands comprises one or more -L-Y-CO2H group of a charged form thereof.
  • Y is alkylene.
  • Y is C6-C30 alkylene, for example, C6-C28 alkylene, C6-C26 alkylene, C6-C24 alkylene, C6-C22 alkylene, C6-C20 alkylene, C8-C30 alkylene, C10-C30 alkylene, C10-C28 alkylene, C12-C28 alkylene, C14-C28 alkylene, C16- C28 alkylene, or C16-C22 alkylene. In some embodiments, Y is C16-C22 alkylene.
  • L when Y is alkylene, then L comprises -O(CH2)a(OCH2CH2)bNH-, wherein a is 1-6, and b is 0-5. In some embodiments, a is 1 and b is 2. In some embodiments, a is 1 and b is 0. In some embodiments, a is 1 and b is 1. In some embodiments, Y is alkenylene.
  • Y is C 6 -C 30 alkenylene, for example, C 6 -C 28 alkenylene, C 6 -C 26 alkenylene, C 6 -C 24 alkenylene, C 6 -C 22 alkenylene, C 6 - C20 alkenylene, C8-C30 alkenylene, C10-C30 alkenylene, C10-C28 alkenylene, C12-C28 alkenylene, C 14 -C 28 alkenylene, C 16 -C 28 alkenylene, or C 16 -C 22 alkenylene.
  • the alkenylene comprises from 1-6 olefinic bonds, including 1-5 olefinic bonds, 1-4 olefinic bonds, 1-3 olefinic bonds, or 1-2 olefinic bonds.
  • Y is alkynylene.
  • Y is C6-C30 alkynylene, for example, C6-C28 alkynylene, C6-C26 alkynylene, C6-C24 alkynylene, C6-C22 alkynylene, C6- C20 alkynylene, C8-C30 alkynylene, C10-C30 alkynylene, C10-C28 alkynylene, C12-C28 alkynylene, C14-C28 alkynylene, C16-C28 alkynylene, or C16-C22 alkynylene.
  • the alkynylene comprises from 1-6 olefinic bonds, including 1-5 olefinic bonds, 1-4 olefinic bonds, 1-3 olefinic bonds, or 1-2 olefinic bonds.
  • each ligand comprises a linker (L), wherein L is conjugated the one or more alkylene-CO 2 H, alkenylene-CO 2 H, or alkynylene-CO 2 H, or a charged form thereof.
  • the alkylene-CO 2 H, alkenylene-CO 2 H, or alkynylene-CO 2 H is C 5-24 alkylene-CO 2 H, C 5-24 alkenylene-CO 2 H, or C 5-24 alkynylene-CO 2, or a charged form thereof.
  • the alkylene-CO2H, alkenylene-CO2H, or alkynylene-CO2H is C9-24 alkylene-CO2H, C9-24 alkenylene-CO2H, or C9-24 alkynylene-CO2, or a charged form thereof.
  • the alkylene-CO2H, alkenylene-CO2H, or alkynylene-CO2H is FH12501620.1 Attorney Docket: DCY-13025 C 15-22 alkylene-CO 2 H, C 15-22 alkenylene-CO 2 H, or C 15-22 or alkynylene-CO 2 H, or a charged form thereof.
  • L comprises a bivalent or trivalent C 1 -C 50 alkylene, wherein 1- 25 methylene groups are optionally and independently replaced by -N(H)-, -N(C 1 -C 4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, - S(O)2N(cycloalkyl)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), -C(O)N(cycloalkyl), aryl, heteroaryl, cycl
  • the 1-25 methylene groups are optionally and independently replaced by -C(O)-, -N(H)-, -N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -O-, or heteroaryl, or combinations thereof.
  • at least one methylene group is replaced by a 5-12 membered heteroaryl.
  • the 5-12 membered heteroaryl comprises 1-5 heteroatoms (e.g., 1, 2, 3, 4, or 5, including any values or ranges therebetween) selected from N, O, or S.
  • the 1-25 methylene groups are optionally and independently replaced by a 5-8 membered heteroaryl with up to 1-3 heteroatoms (e.g., 1, 2, or 3, including any values or ranges therebetween) selected from N, O, or S.
  • the heteroaryl is a 5 membered heteroaryl with up to 1-3 heteroatoms selected from N, O, or S.
  • heteroaryl is triazolyl, pyrrolyl, pyrazolyl, imidazolyl, isozazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl.
  • the heteroaryl is a triazolyl. In some embodiments, the triazolyl is .
  • the ligand is L-Y-CO 2 H. In some embodiments, the ligand M is absent, or a bonding group (as described herein) such as NRC(O), or heteroaryl, wherein R is H or alkyl.
  • a is 1, 2, 3, 4, 5, or 6;
  • b is 1, 2, 3, 4, 5, 6, 7, or 8;
  • c is 0, 1, 2, 3, 4, 5, or 6;
  • d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
  • -L-Y-CO 2 H is , wherein: M is absent, or a bonding group (as described herein) such as -NRC(O)-, or heteroaryl, wherein R is H or alkyl.
  • c is 0, 1, 2, 3, 4, 5, or 6;
  • d is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
  • M is -NRC(O)-.
  • R is H or methyl.
  • R is H.
  • M is heteroaryl.
  • the heteroaryl is triazolyl. In some embodiments, the triazolyl is . In some embodiments, M is absent. In some embodiments, c is 1. In some embodiments, d is 0 to 12. In some embodiments, d is 4 to 12. In some embodiments, d is 0. In some embodiments, e is 12 to 23. In some embodiments, e is 11, 15, or 21.
  • the antisense strand includes an overhang at the 3’ end of 2 to 6 nucleotides (e.g., 2, 3, 4, 5, or 6 nucleotides). In some embodiments, the antisense strand comprises at least one phosphorothioate linkage.
  • the antisense strand comprises one or more modified nucleotides.
  • modified nucleotide comprises a modified sugar.
  • the modified sugar comprises a 2’-OMe group.
  • the modified sugar comprises a 2’-F substituent.
  • the antisense strand comprises up to 4 contiguous nucleotides with a 2’-F modified sugar.
  • the antisense strand comprises a 4’-O-monomethylphosphonate-2’-O-methyl modified nucleotide.
  • the ligand is conjugated to a ribose of the sense strand at the 3’- or 5’-position.
  • At least one ligand is conjugated to a ribose of the sense strand at the 2’-position.
  • FH12501620.1 Attorney Docket: DCY-13025 Formulas
  • the present disclosure provides an oligonucleotide-ligand conjugate of Formula (AI): (AI) or a pharmaceutically acceptable salt or charged form thereof, wherein: A and A’ are each independently H or one or more nucleotide; B is a nucleobase; Z is O or S; M is absent, or a moiety formed by conjugating the carboxyl containing component to the remainder of the ligand, such as NRC(O), or heteroaryl, wherein R is H or alkyl.
  • the oligonucleotide-ligand conjugate of Formula (AI) has a structure of Formula (AII): FH12501620.1 Attorney Docket: DCY-13025 (AII), or a pharmaceutically acceptable salt or charged form thereof, wherein A, A’, B, Z, a, b, d, e, and f are defined above in Formula (AI).
  • the oligonucleotide-ligand conjugate of Formula (AI) has a structure of Formula (AIII): (AIII), or a pharmaceutically acceptable salt or charged form thereof, wherein A, A’, B, Z, a, b, d, e, and f are defined above in Formula (AI).
  • the oligonucleotide-ligand conjugate of Formula (AI) or (AIII) has a structure of Formula (AIV): (AIV), or a pharmaceutically acceptable salt or charged form thereof, wherein A, A’, B, Z, a, b, e, and f are defined above in Formula (AI).
  • the oligonucleotide-ligand conjugate of Formula (AI) has a structure of Formula (AV): FH12501620.1 Attorney Docket: DCY-13025 (AV), or a pharmaceutically acceptable salt or charged form thereof, wherein A, A’, B, Z, a, b, d, and f are defined above in Formula (AI), and wherein e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23.
  • the present disclosure provides an oligonucleotide-ligand conjugate of Formula (BI) (BI), or a pharmaceutically acceptable salt or charged form thereof, wherein: A is one or more nucleotide; B is a nucleobase; Z 1 and Z 2 are each independently O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl.
  • a is 1, 2, 3, 4, or 5;
  • c is 0, 1, 2, 3, 4, 5, or 6;
  • d1 and d2 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or e is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 23; and f is 0, 1, 2, 3, 4, 5, or 6.
  • the present disclosure provides an oligonucleotide-ligand conjugate of Formula (BII): FH12501620.1 Attorney Docket: DCY-13025 (BII), or a salt or charged form thereof, wherein B, A, M, a, c, d1, d2, e, and f are defined above in Formula (BI), and wherein Z is O or S.
  • Formula (BII) FH12501620.1 Attorney Docket: DCY-13025 (BII), or a salt or charged form thereof, wherein B, A, M, a, c, d1, d2, e, and f are defined above in Formula (BI), and wherein Z is O or S.
  • the present disclosure provides an oligonucleotide-ligand conjugate of Formula (CI): (CI), or a pharmaceutically acceptable salt or charged form thereof, wherein: A is one or more nucleotide; B is a nucleobase; Z 1 and Z 2 are each independently O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl.
  • CI oligonucleotide-ligand conjugate of Formula (CI): (CI), or a pharmaceutically acceptable salt or charged form thereof, wherein: A is one or more nucleotide; B is a nucleobase; Z 1 and Z 2 are each independently O or S; M is absent, NRC(O), or heteroaryl, wherein R is H or alkyl.
  • the present disclosure provides an oligonucleotide-ligand conjugate of Formula (CII): FH12501620.1 Attorney Docket: DCY-13025 (CII), or a salt or charged form thereof, wherein B, A, M, a, c, d1, d2, e, and f are defined above in Formula (CI), and wherein Z is O or S.
  • M is any suitable group (e.g., a reaction product) that couples the hydrocarbon bearing one or more carboxyl groups to the linker (L).
  • M is -NRC(O)-.
  • R is H.
  • R is alkyl.
  • R is C1-6 alkyl.
  • R is methyl.
  • R is ethyl.
  • M is heteroaryl.
  • M is a 5-12 membered heteroaryl.
  • M is a 5-12 membered heteroaryl containing 1-5 heteroatoms (e.g., 1, 2, 3, 4, or 5, including any ranges therebetween) selected from N, O, or S.
  • M is a 5-8 membered heteroaryl containing 1-3 heteroatoms (e.g., 1, 2, or 3, including any values or ranges therebetween) selected from N, O, or S.
  • M is a 5-7 membered heteroaryl containing 1-3 heteroatoms selected from N, O, or S.
  • M is triazolyl, pyrrolyl, pyrazolyl, imidazolyl, isozazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl.
  • M is triazolyl.
  • M is absent.
  • M groups include: , , , B
  • B In some embodiments of the oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), B is a natural or non-natural base. In some embodiments, the base is FH12501620.1 Attorney Docket: DCY-13025 some embodiments, B is . In some embodiments, B is .
  • Z In some embodiments of the oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), Z is O. In some embodiments, Z is S. a, b, c, d, e, f In some embodiments of the oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5.
  • a is 6. In some embodiments, a is 1 or 2. In some embodiments of the oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), or (AV), b is 1. In some embodiments, b is 2. In some embodiments, b is 3. In some embodiments, b is 4. In some embodiments, b is 5. In some embodiments, b is 6. In some embodiments, b is 1 or 2. In some embodiments, c is 0. In some embodiments, c is 1. In some embodiments, c is 2. In some embodiments, c is 3. In some embodiments, c is 4. In some embodiments, c is 5. In some embodiments, c is 6.
  • d is 0. In some embodiments, d is 1. In some embodiments, d is 2. In some embodiments, d is 3. In some embodiments, d is 4. In some embodiments, d is 5. In some embodiments, d is 6. In some embodiments, d is 7. In some embodiments, d is 8. In some embodiments, d is 9. In some embodiments, d is 10. In some embodiments, d is 11. In FH12501620.1 Attorney Docket: DCY-13025 some embodiments, d is 12. In some embodiments, d is 13. In some embodiments, d is 14.
  • d is 15. In some embodiments, d is 16. In some embodiments, d is 17. In some embodiments, d is 18. In some embodiments, d is 19. In some embodiments, d is 20. In some embodiments, d is 0, 4, or 12. In some embodiments, d is 0 or 12. In some embodiments of the oligonucleotide-ligand conjugate of Formulas (BI), (BII), (CI), or (CII), d1 is 0. In some embodiments, d1 is 1. In some embodiments, d1 is 2. In some embodiments, d1 is 3. In some embodiments, d1 is 4. In some embodiments, d1 is 5. In some embodiments, d1 is 6. In some embodiments, d1 is 7.
  • d1 is 8. In some embodiments, d1 is 9. In some embodiments, d1 is 10. In some embodiments, d1 is 11. In some embodiments, d1 is 12. In some embodiments, d1 is 13. In some embodiments, d1 is 14. In some embodiments, d1 is 15. In some embodiments, d1 is 16. In some embodiments, d1 is 17. In some embodiments, d1 is 18. In some embodiments, d1 is 19. In some embodiments, d1 is 20. In some embodiments of the oligonucleotide-ligand conjugate of Formulas (BI), (BII), (CI), or (CII), d 2 is 0. In some embodiments, d 2 is 1.
  • d 2 is 2. In some embodiments, d 2 is 3. In some embodiments, d 2 is 4. In some embodiments, d 2 is 5. In some embodiments, d2 is 6. In some embodiments, d2 is 7. In some embodiments, d2 is 8. In some embodiments, d 2 is 9. In some embodiments, d 2 is 10. In some embodiments, d 2 is 11. In some embodiments, d2 is 12. In some embodiments, d2 is 13. In some embodiments, d2 is 14. In some embodiments, d2 is 15. In some embodiments, d2 is 16. In some embodiments, d2 is 17. In some embodiments, d2 is 18. In some embodiments, d2 is 19. In some embodiments, d2 is 20.
  • e is 5. In some embodiments, e is 6. In some embodiments, e is 7. In some embodiments, e is 8. In some embodiments, e is 9. In some embodiments, e is 10. In some embodiments, e is 11. In some embodiments, e is 12. In some embodiments, e is 13. In some embodiments, e is 14. In some embodiments, e is 15. In some embodiments, e is 16. In some embodiments, e is 17. In some embodiments, e is 18. In some embodiments, e is 19.
  • e is 20. In some embodiments, e is 21. In some embodiments, e is 22. In some embodiments, e is 23. In some embodiments, e is 11 or 22. In some embodiments of the oligonucleotide-ligand, e is 7, 15, or 21. In some embodiments of the oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), e is 11, 15, or 21.
  • f is 0.
  • f is 1.
  • f is 2.
  • f is 3.
  • f is 4.
  • f is 5.
  • f is 6.
  • f is 0, 1, or 2.
  • a and A ⁇ In some embodiments of the oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), A is H. In some embodiments of the oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), or (AV) A ⁇ is H. In some embodiments, A is 13 to 40 nucleotides in length. In some embodiments, A is 13 to 36 nucleotides in length. In some embodiments, A is 12 to 29 nucleotides in length. In some embodiments, A is 15 to 20 nucleotides in length.
  • A is 13 nucleotides in length. In some embodiments, A is 14 nucleotides in length. In some embodiments, A is 15 nucleotides in length. In some embodiments, A is 16 nucleotides in length. In some embodiments, A is 17 nucleotides in length. In some embodiments, A is 18 nucleotides in length. In some embodiments, A is 19 nucleotides in length. In some embodiments, A is 20 nucleotides in length. In some embodiments, A is 21 nucleotides in length. In some embodiments, A is 22 nucleotides in length. In some embodiments, A is 14 nucleotides in length. In some embodiments, A is 23 nucleotides in length.
  • A is 24 nucleotides in length. In some embodiments, A is 25 nucleotides in length. In some embodiments, A is 26 nucleotides in length. In some embodiments, A is 27 nucleotides in length. In some embodiments, A is 28 nucleotides in length. In some embodiments, A is 29 nucleotides in length. In some embodiments, A is 30 nucleotides in length. In some embodiments, A is 31 nucleotides in length. In some embodiments, A is 32 nucleotides in length. In some embodiments, A is 33 nucleotides in length. In some embodiments, A is 34 nucleotides in length. In some embodiments, A is 35 nucleotides in length.
  • A is 36 nucleotides in length. In some embodiments, A is 37 nucleotides in length. In some embodiments, A is 38 nucleotides in length. In some embodiments, A is 39 nucleotides in length. In some embodiments, A is 40 nucleotides in length. In some embodiments, A ⁇ is 13 to 40 nucleotides in length. In some embodiments, A ⁇ is 13 to 36 nucleotides in length. In some embodiments, A ⁇ is 15 to 20 nucleotides in length. In some embodiments, A ⁇ is 13 nucleotides in length. In some embodiments, A ⁇ is 14 nucleotides in length. In some embodiments, A ⁇ is 15 nucleotides in length.
  • a ⁇ is FH12501620.1 Attorney Docket: DCY-13025 16 nucleotides in length. In some embodiments, A ⁇ is 17 nucleotides in length. In some embodiments, A ⁇ is 18 nucleotides in length. In some embodiments, A ⁇ is 19 nucleotides in length. In some embodiments, A is 20 nucleotides in length. In some embodiments, A ⁇ is 21 nucleotides in length. In some embodiments, A ⁇ is 22 nucleotides in length. In some embodiments, A ⁇ is 14 nucleotides in length. In some embodiments, A ⁇ is 23 nucleotides in length. In some embodiments, A ⁇ is 24 nucleotides in length.
  • a ⁇ is 25 nucleotides in length. In some embodiments, A ⁇ is 26 nucleotides in length. In some embodiments, A ⁇ is 27 nucleotides in length. In some embodiments, A ⁇ is 28 nucleotides in length. In some embodiments, A ⁇ is 29 nucleotides in length. In some embodiments, A ⁇ is 30 nucleotides in length. In some embodiments, A ⁇ is 31 nucleotides in length. In some embodiments, A ⁇ is 32 nucleotides in length. In some embodiments, A ⁇ is 33 nucleotides in length. In some embodiments, A ⁇ is 34 nucleotides in length. In some embodiments, A ⁇ is 35 nucleotides in length.
  • a ⁇ is 36 nucleotides in length. In some embodiments, A ⁇ is 37 nucleotides in length. In some embodiments, A ⁇ is 38 nucleotides in length. In some embodiments, A ⁇ is 39 nucleotides in length. In some embodiments, A ⁇ is 40 nucleotides in length. In some embodiments, A is 12-36 nucleotides in length and A ⁇ is 1-10 nucleotides in length. In some embodiments, A is 12-29 nucleotides in length and A ⁇ is 1-10 nucleotides in length. In some embodiments, A is 1-10 nucleotides in length and A ⁇ is 12-29 nucleotides in length.
  • A is 13 to 40 nucleotides in length and A ⁇ is H. In some embodiments, A is 13 to 20 nucleotides in length and A ⁇ is H. In some embodiments, A is 18 to 30 nucleotides in length and A ⁇ is H. In some embodiments, A is 27 nucleotides in length and A ⁇ is 8 nucleotides in length. In some embodiments, A is 28 nucleotides in length and A ⁇ is 8 nucleotides in length. In some embodiments, A is H and A ⁇ is 12 to 40 nucleotides in length. In some embodiments, A is H and A ⁇ is 36 nucleotides in length. In some embodiments, A is H and A ⁇ is 19 nucleotides in length.
  • an RNAi oligonucleotide comprising a sense strand and an antisense strand comprises an oligonucleotide-ligand conjugate of any one Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprising a sense strand and an antisense strand comprises at least one oligonucleotide-ligand conjugate of any one of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprising a sense strand and an antisense strand comprises at least two oligonucleotide-ligand conjugates of any one of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AI), wherein A is H and A ⁇ is 19 nucleotides, and wherein the antisense strand is 15 to 30 nucleotides in length.
  • AI oligonucleotide-ligand conjugate of Formula
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AI), wherein A is H and A ⁇ is 19 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AI), wherein A is 27 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 15 to 30 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AI), wherein A is 27 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AI), wherein A is 28 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 15 to 30 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AI), wherein A is 28 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AII), wherein A is H and A ⁇ is 19 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AII), wherein A is 27 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AII), wherein A is 28 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AIII), wherein A is H and A ⁇ is 19 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and FH12501620.1 Attorney Docket: DCY-13025 antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AIII), wherein A is 27 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AIII), wherein A is 28 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AIV), wherein A is H and A ⁇ is 19 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AIV), wherein A is 27 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AIV), wherein A is 28 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AV), wherein A is H and A ⁇ is 19 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AV), wherein A is 27 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of Formula (AV), wherein A is 28 nucleotides and A ⁇ is 8 nucleotides, and wherein the antisense strand is 22 nucleotides in length.
  • the RNAi oligonucleotide comprises a duplex region.
  • the duplex region includes one or more phosphorothioate linkages (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more phosphorothioate linkages).
  • the duplex region includes two phosphorothioate linkages.
  • the duplex region includes three phosphorothioate linkages. In some embodiments, the duplex region includes four phosphorothioate linkages. In some embodiments, the duplex region includes two or more phosphorothioate linkages, and two phosphorothioate linkages are adjacent to each other. In some embodiments, the duplex region includes two phosphorothioate linkages, and FH12501620.1 Attorney Docket: DCY-13025 the two phosphorothioate linkages are adjacent to each other. In some embodiments, the duplex region comprises 20 to 30 base pairs, including 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, including any values or ranges therebetween.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A and A ⁇ comprise at least 14 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 14 nucleotides of A and A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A comprises at least 14 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 14 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A ⁇ comprises at least 14 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 14 nucleotides of A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A and A ⁇ comprise at least 15 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 15 nucleotides of A and A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A comprises at least 15 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 15 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A ⁇ comprises at least 15 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 15 nucleotides of A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A and A ⁇ comprise at least 16 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 16 nucleotides FH12501620.1 Attorney Docket: DCY-13025 of A and A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A comprises at least 16 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 16 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A ⁇ comprises at least 16 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 16 nucleotides of A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A and A ⁇ comprise at least 17 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 17 nucleotides of A and A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A comprises at least 17 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 17 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A ⁇ comprises at least 17 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 17 nucleotides of A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A and A ⁇ comprise at least 18 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 18 nucleotides of A and A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A comprises at least 18 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 18 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of FH12501620.1 Attorney Docket: DCY-13025 Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A ⁇ comprises at least 18 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 18 nucleotides of A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A and A ⁇ comprise at least 19 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 19 nucleotides of A and A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A comprises at least 19 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 19 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (AI), (AII), (AIII), (AIV), and (AV), wherein A ⁇ comprises at least 19 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 19 nucleotides of A ⁇ form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (BI), (BII), (CI), and (CII)
  • A comprises at least 14 nucleotides
  • the oligonucleotide-ligand conjugate and the at least 14 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide- ligand conjugate of any of Formula (BI), (BII), (CI), and (CII)
  • A comprises at least 15 nucleotides
  • the oligonucleotide-ligand conjugate and the at least 15 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (BI), (BII), (CI), and (CII)
  • A comprises at least 16 nucleotides
  • the oligonucleotide-ligand conjugate and the at least 16 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand
  • the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (BI), (BII), (CI), and (CII)
  • A comprises at least 17 nucleotides
  • the oligonucleotide-ligand conjugate and the at least 17 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense FH12501620.1 Attorney Docket: DCY-13025 strand and an antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (BI), (BII), (CI), and (CII), wherein A comprises at least 18 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 18 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of any of Formula (BI), (BII), (CI), and (CII), wherein A comprises at least 19 nucleotides, and wherein the oligonucleotide-ligand conjugate and the at least 19 nucleotides of A form a duplex region with the antisense strand.
  • the RNAi oligonucleotide comprises a loop region.
  • the RNAi oligonucleotide comprises a loop region, wherein the loop comprises an oligonucleotide-ligand conjugate of any of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • the RNAi oligonucleotide comprises a loop region, wherein the loop is a tetraloop comprising an oligonucleotide-ligand conjugate of any of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an oligonucleotide-ligand conjugate of any of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII) is position 1 of the tetraloop.
  • an oligonucleotide-ligand conjugate of any of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII) is position 2 of the tetraloop. In some embodiments, an oligonucleotide- ligand conjugate of any of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII) is position 3 of the tetraloop.
  • an oligonucleotide-ligand conjugate of any of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII) is position 4 of the tetraloop.
  • the tetraloop region comprises a 5’- GAAA-3’ sequence.
  • an RNAi oligonucleotide comprising an oligonucleotide- ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII) comprises one or more modified nucleotides, e.g., as described herein.
  • the modified nucleotide comprises a modified sugar.
  • the modified sugar comprises a 2’-OMe substituent.
  • the modified sugar comprises a 2’-F substituent.
  • the modified nucleotide comprises a modified nucleobase.
  • the modified nucleotide is a 4’-O- monomethylphosphonate-2’-O-methyl modified nucleotide.
  • oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein A is at least one nucleotide, one or more nucleotides of A is a modified nucleotide.
  • FH12501620.1 Attorney Docket: DCY-13025 oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein A is 13 to 40 nucleotides, one or more nucleotides of A is a modified nucleotide.
  • oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII) wherein A is 13 to 40 nucleotides, one or more nucleotides of A comprises a 2’-OMe substituent.
  • oligonucleotide-ligand conjugate of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII) wherein A is 13 to 40 nucleotides, every nucleotide of A is a modified nucleotide.
  • one or more nucleotides of A ⁇ is a modified nucleotide.
  • a ⁇ is 13 to 40 nucleotides, every nucleotide of A ⁇ is a modified nucleotide.
  • the conjugate further comprises one or more ligands having the structure -L-Y-(CO 2 H) n as described herein.
  • the ligand is conjugated to a ribose of the sense strand at the 2’-position.
  • ligand is conjugated to a ribose of the sense strand at the 3’- or 5’-position.
  • FH12501620.1 Attorney Docket: DCY-13025
  • the present disclosure provides an oligonucleotide-ligand conjugate comprises two or more of (i), (ii), or (iii): (i) Formula (AI), (AII), (AIII), (AIV), or (AV); (ii) Formula (BI) or (BII); and (iii) Formula (CI) or (CII).
  • RNAi Oligonucleotide Targeting Sequences In some embodiments, the RNAi oligonucleotides provided by the disclosure comprise a targeting sequence.
  • the 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 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 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 FH12501620.1 Attorney Docket: DCY-13025 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 RNAi oligonucleotides herein comprise a targeting sequence that is fully complementary to a target sequence comprising a target mRNA. In some embodiments, the 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 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 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. In some embodiments, the 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 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. In some embodiments, the 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.
  • the 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 an 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.
  • a targeting sequence of an 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. In some embodiments, a targeting sequence of an 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 an 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 an 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 an 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.
  • an 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 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 RNAi oligonucleotide to inhibit or reduce target gene expression is maintained.
  • the RNAi oligonucleotide comprises a targeting sequence having 1 mismatch with the corresponding target sequence.
  • the RNAi oligonucleotide comprises a targeting sequence having 2 mismatches with the corresponding target sequence.
  • the RNAi oligonucleotide comprises a targeting sequence having 3 mismatches with the corresponding target sequence. In some embodiments, the RNAi oligonucleotide comprises a targeting sequence having 4 mismatches with the corresponding target sequence. In some embodiments, the RNAi oligonucleotide comprises a targeting sequence having 5 mismatches with the corresponding target sequence.
  • the 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.
  • mismatches e.g., 2, 3, 4, 5 or more mismatches
  • the 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.
  • mismatches e.g., 2, 3, 4, 5 or more mismatches
  • Types of Oligonucleotides A variety of RNAi oligonucleotide types and/or structures are useful for reducing target gene expression in the methods herein.
  • 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 RNAi oligonucleotides herein inhibit target gene expression by engaging with RNA interference (RNAi) pathways upstream or downstream of Dicer involvement.
  • 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).
  • oligonucleotides 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). Further work produced 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 Intl. 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.
  • double-stranded nucleic acids of 19-23 nucleotides in length capable of reducing expression of a target mRNA are produced.
  • the RNAi oligonucleotide has an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3 ⁇ end of the sense strand.
  • the RNAi oligonucleotide (e.g., siRNA conjugate) comprises a 21-nucleotide guide strand that is antisense to a target mRNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3 ⁇ ends.
  • 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.
  • 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. In some embodiments, the 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 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.
  • an 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).
  • 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.), R OYAL S OCIETY OF C HEMISTRY , 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. (2010) M ETHODS M OL .
  • siRNAs see, e.g., Nucleic Acids in Chemistry and Biology, Blackburn (ed.), R OYAL S OCIETY OF C HEMISTRY , 2006
  • shRNAs e.g., having 19 bp or shorter stems; see, e.g., Moore et al. (2010) M ETHODS M OL .
  • B IOL .629:141-58 blunt siRNAs (e.g., of 19 bps in FH12501620.1 Attorney Docket: DCY-13025 length; see, e.g., Kraynack & Baker (2006) RNA 12:163-76), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al. (2008) NAT. BIOTECHNOL.26:1379-82), asymmetric shorter-duplex siRNA (see, e.g., Chang et al. (2009) MOL. THER.17:725-32), fork siRNAs (see, e.g., Hohjoh (2004) FEBS Lett.
  • aiRNA see, e.g., Sun et al. (2008) NAT. BIOTECHNOL.26:1379-82
  • asymmetric shorter-duplex siRNA see, e.g., Chang et al. (2009) MOL. THER.17:725-32
  • RNA small internally segmented interfering RNA
  • siRNA small internally segmented interfering RNA
  • miRNA microRNA
  • shRNA short hairpin RNA
  • siRNA siRNA
  • an antisense strand of an 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, the antisense strand is referred to as a guide strand.
  • RISC RNA-induced silencing complex
  • 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).
  • an 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.
  • an RNAi oligonucleotide herein comprises an antisense strand of 15 to 30 nucleotides in length.
  • an antisense strand of any one of the 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.
  • an RNAi oligonucleotide comprises an antisense strand of 19-23 nucleotides in length.
  • an RNAi oligonucleotide comprises an antisense strand of 19 nucleotides in length.
  • an RNAi oligonucleotide comprises an antisense strand of 20 nucleotides in length. In some embodiments, an RNAi oligonucleotide comprises an antisense 76 FH12501620.1 Attorney Docket: DCY-13025 strand of 21 nucleotides in length. In some embodiments, an RNAi oligonucleotide comprises an antisense strand of 22 nucleotides in length. In some embodiments, an RNAi oligonucleotide comprises an antisense strand of 23 nucleotides in length.
  • an 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).
  • an 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).
  • an 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.
  • an RNAi oligonucleotide herein comprises a sense strand 15 to 50 nucleotides in length.
  • an RNAi oligonucleotide herein comprises a sense strand 18 to 38 nucleotides in length.
  • an 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.
  • an RNAi oligonucleotide herein comprises a sense strand of 12-21 nucleotides in length.
  • an RNAi oligonucleotide herein comprises a sense strand of 12 nucleotides in length.
  • an RNAi oligonucleotide herein comprises a sense strand of 13 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 14 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 15 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 16 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 17 nucleotides in length.
  • an RNAi oligonucleotide herein comprises a sense strand of 18 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 19 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 20 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 21 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 22 nucleotides in length.
  • an RNAi FH12501620.1 Attorney Docket: DCY-13025 oligonucleotide herein comprises a sense strand of 23 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 24 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 25 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 26 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 27 nucleotides in length.
  • an RNAi oligonucleotide herein comprises a sense strand of 28 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 29 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 30 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 31 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 32 nucleotides in length.
  • an RNAi oligonucleotide herein comprises a sense strand of 33 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 34 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 35 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 36 nucleotides in length. In some embodiments, an RNAi oligonucleotide herein comprises a sense strand of 37 nucleotides in length.
  • an RNAi oligonucleotide herein comprises a sense strand of 38 nucleotides in length.
  • a sense strand comprises a stem-loop structure at its 3 ⁇ end.
  • a sense strand comprises a stem-loop structure at its 5 ⁇ end.
  • the stem-loop is formed by intrastrand base pairing.
  • a sense strand comprises a stem-loop structure at its 5 ⁇ end.
  • a stem is a duplex of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length.
  • the stem of the stem-loop comprises a duplex of 1 nucleotide in length.
  • the stem of the stem-loop comprises a duplex of 2 nucleotides in length. In some embodiments, 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.
  • the stem of the stem-loop comprises a duplex of 8 nucleotides in length. In some embodiments, 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 FH12501620.1 Attorney Docket: DCY-13025 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.
  • the stem of the stem-loop comprises a duplex of 14 nucleotides in length.
  • a stem-loop provides the 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 extrahepatic tissue), inhibition of target gene expression, and/or delivery to a target cell, tissue, or organ (e.g., extrahepatic tissue), or a combination thereof.
  • a target mRNA e.g., a target mRNA expressed in extrahepatic tissue
  • a target cell, tissue, or organ e.g., extrahepatic tissue
  • the stem-loop itself or modification(s) to the stem-loop do not substantially affect the inherent gene expression inhibition activity of the RNAi oligonucleotide, but facilitates, improves, or increases stability (e.g., provides protection against degradation) and/or delivery of the RNAi oligonucleotide to a target cell, tissue, or organ (e.g., extrahepatic tissue ).
  • an 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 S1 is complementary to S2, and in which L forms a single-stranded loop between S1 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.
  • the loop (L) is 4 nucleotides in length.
  • the tetraloop comprises the sequence 5’-GAAA-3’.
  • 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: 11). In some embodiments, a loop (L) of a stem-loop having the structure S1-L-S2 as described above is a triloop. In some embodiments, 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.
  • FH12501620.1 Attorney Docket: DCY-13025
  • 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.
  • 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).
  • 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.
  • a duplex formed between a sense and antisense strand is 10-18 base pairs in length.
  • a duplex formed between a sense and antisense strand is 15- 30 base pairs in length.
  • a duplex formed between a sense and antisense strand is 17-21 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. In some embodiments, a duplex formed between a sense and antisense strand is 17 base pairs in length.
  • 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. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands.
  • a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.
  • FH12501620.1 Attorney Docket: DCY-13025 Oligonucleotide Ends
  • an 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.
  • an RNAi oligonucleotide herein has one 5’end that is thermodynamically less stable compared to the other 5’ end.
  • an asymmetric RNAi oligonucleotide 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 to twenty 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 3’ overhang is about one to nineteen, one to eighteen, one to seventeen, one to sixteen, one to fifteen, one to fourteen, one to thirteen, one to twelve, one to eleven, one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, or about one to two nucleotides in length.
  • the 3’-overhang is nucleotide in length.
  • the 3’-overhang is two nucleotides in length.
  • the 3’-overhang is three nucleotides in length.
  • the 3’-overhang is four nucleotides in length.
  • the 3’-overhang is five nucleotides in length. In some embodiments, the 3’- overhang is six nucleotides in length. In some embodiments, the 3’-overhang is seven nucleotides in length. In some embodiments, the 3’-overhang is eight nucleotides in length. In some embodiments, the 3’-overhang is nine nucleotides in length. In some embodiments, the 3’-overhang is ten nucleotides in length. In some embodiments, the 3’-overhang is eleven nucleotides in length. In some embodiments, the 3’-overhang is twelve nucleotides in length. In some embodiments, the 3’-overhang is thirteen nucleotides in length.
  • the 3’-overhang is fourteen nucleotides in length. In some embodiments, the 3’-overhang is fifteen nucleotides in length. In some embodiments, the 3’-overhang is sixteen nucleotides in length. In some embodiments, the 3’-overhang is seventeen nucleotides in length. In some embodiments, the 3’-overhang is eighteen nucleotides in length. In some embodiments, the 3’-overhang is nineteen nucleotides in length. In some embodiments, the 3’-overhang is twenty nucleotides in length.
  • an oligonucleotide for RNAi has a two 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, FH12501620.1 Attorney Docket: DCY-13025 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.
  • an oligonucleotide herein comprises a sense strand and an antisense strand, wherein the sense strand comprises a 5’- overhang comprising one or more nucleotides.
  • 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 to twenty 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 to nineteen, one to eighteen, one to seventeen, one to sixteen, one to fifteen, one to fourteen, one to thirteen, one to twelve, one to eleven, one to ten, one to nine, one to eight, one to seven, one to six, one to five, one to four, one to three, or about one to two nucleotides in length.
  • the 5’-overhang is nucleotide in length.
  • the 5’-overhang is two nucleotides in length.
  • the 5’-overhang is three nucleotides in length.
  • the 5’-overhang is four nucleotides in length.
  • the 5’-overhang is five nucleotides in length. In some embodiments, the 5’- overhang is six nucleotides in length. In some embodiments, the 5’-overhang is seven nucleotides in length. In some embodiments, the 5’-overhang is eight nucleotides in length. In some embodiments, the 5’-overhang is nine nucleotides in length. In some embodiments, the 5’-overhang is ten nucleotides in length. In some embodiments, the 5’-overhang is eleven nucleotides in length. In some embodiments, the 5’-overhang is twelve nucleotides in length. In some embodiments, the 5’-overhang is thirteen nucleotides in length.
  • an RNAi oligonucleotide 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 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 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 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 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 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.
  • the overhang is AA.
  • the overhang is AG.
  • the overhang is GA.
  • the two terminal overhang nucleotides are GG.
  • 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.
  • FH12501620.1 Attorney Docket: DCY-13025
  • one or more (e.g., 2, 3, 4, 5, 6) modified internucleotide linkages are provided between terminal nucleotides of the 3’ end or 5’ end of a sense and/or antisense strand.
  • modified internucleotide linkages are provided between overhang nucleotides at the 3’ end or 5’ end of a sense and/or antisense strand.
  • Oligonucleotide Modifications in some embodiments, an RNAi oligonucleotide disclosed herein comprises one or more modifications. Oligonucleotides (e.g., RNAi oligonucleotides) may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-pairing properties, RNA distribution and cellular uptake and other features relevant to therapeutic research use. In some embodiments, the modification is a modified sugar.
  • the modification is a 5’-terminal phosphate group. In some embodiments, the modification is a modified internucleoside linkage. In some embodiments, the modification is a modified base. In some embodiments, an oligonucleotide described herein can comprise any one of the modifications described herein or any combination thereof. For example, in some embodiments, 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 ligand (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 FH12501620.1 Attorney Docket: DCY-13025 characteristics (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).
  • DCY-13025 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 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA) or 2 ⁇ -deoxy-2 ⁇ - fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • the modification is 2 ⁇ -F, 2 ⁇ - OMe or 2 ⁇ -MOE.
  • 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 1 ⁇ -carbon or 4 ⁇ -carbon of the sugar, or a 2 ⁇ -oxygen is linked to the 1 ⁇ -carbon or 4 ⁇ - 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.
  • an 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 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 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). In some embodiments, all the nucleotides of the sense strand of the RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the antisense strand of the RNAi oligonucleotide are modified. In some embodiments, all the nucleotides of the 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 ⁇ -OMe, 2 ⁇ - MOE, and 2 ⁇ -deoxy-2 ⁇ -fluoro- ⁇ -d-arabinonucleic acid).
  • the disclosure provides RNAi oligonucleotides having different modification patterns.
  • the modified RNAi oligonucleotides comprise a sense strand sequence having a modification pattern as set forth in the Examples and Sequence FH12501620.1 Attorney Docket: DCY-13025 Listing and an antisense strand having a modification pattern as set forth in the Examples and Sequence Listing.
  • an RNAi oligonucleotide disclosed herein comprises an antisense strand having nucleotides that are modified with 2 ⁇ -F. In some embodiments, an RNAi oligonucleotide disclosed herein comprises an antisense strand comprises nucleotides that are modified with 2 ⁇ -F and 2 ⁇ -OMe. In some embodiments, an RNAi oligonucleotide disclosed herein comprises a sense strand having nucleotides that are modified with 2 ⁇ -F. In some embodiments, an RNAi oligonucleotide disclosed herein comprises a sense strand comprising nucleotides that are modified with 2 ⁇ -F and 2 ⁇ -OMe.
  • an 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 ligand is not modified with 2’-F or 2’-OMe.
  • 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. In some embodiments, about 11% of the nucleotides of the sense strand comprise a 2-fluoro modification.
  • an RNAi oligonucleotide provided herein 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.
  • one or more nucleotides forming a base pair with a nucleotide at one or more of positions 4, 6, 8, 9, 11, 13, 16, or 18 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 ligand in the sense strand is modified with a 2 ⁇ -OMe.
  • the sugar moiety at each of nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 15, 17, 19, 20, and 21 in the sense strand is modified with a 2 ⁇ - OMe.
  • one or more of positions 3, 5, 8, 10, 12, 13, 15, and 17 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 4, 6, 8, 9, 11, 13, 16, or 18 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 ligand in the sense strand FH12501620.1 Attorney Docket: DCY-13025 is modified with a 2 ⁇ -OMe.
  • the sugar moiety at each of nucleotides at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18, 19, and 20 in the sense strand is modified with a 2 ⁇ - OMe.
  • 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 ligand are modified with a 2’-OMe.
  • at least one internal position (e.g., a position other than the 5’ and 3’ terminal nucleotides) of the sense strand is modified with a 2 ⁇ -F group. In some embodiments, there is no 2 ⁇ -F group at a terminal nucleotide of the sense strand.
  • At least one internal position (e.g., a position other than the 5’ and 3’ terminal nucleotides) of the antisense strand is modified with a 2 ⁇ -F group. In some embodiments, there is no 2 ⁇ -F group at a terminal nucleotide of the antisense strand. In some embodiments, 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.
  • 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, 17, 18, 19, 20, 21, and 22 of the antisense strand are modified with a 2’-OMe. In some embodiments, 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 positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand are modified with a 2’-F.
  • the antisense strand has 12 nucleotides that are modified at the 2’ position of the sugar moiety with a 2’-OMe.
  • the sugar moiety at positions 6, 8, 9, 11, 12, 13, 15, 17, 18, 20, 21, and 22 of the antisense strand are modified with a 2’-OMe.
  • an RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 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 ⁇ -OMe), 2’-O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O- NMA), and 2’-deoxy-2’-fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, FH12501620.1 Attorney Docket: DCY-13025 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 ⁇ -OMe), 2’-O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ - O-NMA), and 2’-deoxy-2’-fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA
  • an 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.
  • an 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 ⁇ -OMe.
  • an 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 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 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ -fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 8-11 modified with 2 ⁇ -F. In some embodiments, an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 4, 6, 9, 11, 13, 14, 16, and 18 modified with 2 ⁇ -F. In some embodiments, an 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, an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1-7 and 12-20 modified with 2’OMe.
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 2-7 and 12-20 modified with 2’OMe. In some embodiments, an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 2-7 and 12-19 modified with 2’OMe. In some FH12501620.1 Attorney Docket: DCY-13025 embodiments, an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 3-7 and 12-20 modified with 2’OMe.
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 1-7 and 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 ⁇ -OMe), 2 ⁇ - O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ - fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 2-7 and 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 ⁇ -OMe), 2 ⁇ - O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ - fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 2-7 and 12-19 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 ⁇ -OMe), 2 ⁇ - O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ - fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 3-7 and 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 ⁇ -OMe), 2 ⁇ - O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ - fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 5, 7, 8, 10, 12, 15, 17, 19, 20, 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 FH12501620.1
  • EA DCY-13025
  • EA 2 ⁇ -O-methyl (2 ⁇ -OMe), 2 ⁇ -O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2- oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ -fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein comprises a sense strand having the sugar moiety at positions 1, 2, 4, 6, 7, 9, 11, 14, 16, 18, 19, and 20of 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 ⁇ -OMe), 2 ⁇ -O- methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ - fluoro- ⁇ -d-arabinonucleic acid (2 ⁇ -FANA).
  • an RNAi oligonucleotide provided herein 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.
  • an RNAi oligonucleotide provided herein 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.
  • an RNAi oligonucleotide provided herein 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 ⁇ -OMe.
  • an RNAi oligonucleotide provided herein 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, FH12501620.1
  • an RNAi oligonucleotide provided herein 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 ⁇ - OMe), 2 ⁇ -O-methoxyethyl (2 ⁇ -MOE), 2 ⁇ -O-[2-(methylamino)-2-oxoethyl] (2 ⁇ -O-NMA), and 2 ⁇ -deoxy-2 ⁇ -fluoro- ⁇ -d-ara
  • the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, or a combination thereof.
  • the 5 ⁇ end of an RNAi oligonucleotide strand is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5 ⁇ -phosphate group (“phosphate mimic”).
  • phosphate mimic a natural 5 ⁇ -phosphate group
  • FH12501620.1 Attorney Docket: DCY-13025
  • an 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., Intl. Patent Application Publication No. WO 2018/045317.
  • an 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.
  • an RNAi oligonucleotide provided herein comprises an antisense strand comprising a phosphorylated nucleotide at the 5’ terminus. In some embodiments, an RNAi oligonucleotide provided herein comprises an antisense strand comprising a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is selected from uridine and adenosine.
  • an RNAi oligonucleotide provided herein comprises an antisense strand comprising a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is uridine. In some embodiments, an RNAi oligonucleotide provided herein comprises an antisense strand comprising a phosphorylated nucleotide at the 5’ terminus, wherein the phosphorylated nucleotide is adenosine. In some embodiments, an RNAi oligonucleotide provided herein comprises an antisense strand comprising a 4 ⁇ -phosphate analog at the 5 ⁇ -terminal nucleotide.
  • an RNAi oligonucleotide provided herein comprises an antisense strand comprising a 4 ⁇ -phosphate analog at the 5 ⁇ -terminal nucleotide, wherein the phosphorylated nucleotide is selected from uridine and adenosine. In some embodiments, an RNAi oligonucleotide provided herein comprises an antisense strand comprising a 4 ⁇ -phosphate analog at the 5 ⁇ -terminal nucleotide, wherein the phosphorylated nucleotide is uridine.
  • an RNAi oligonucleotide provided herein comprises an antisense strand comprising a 4 ⁇ -phosphate analog at the 5 ⁇ -terminal nucleotide, wherein the phosphorylated nucleotide is adenosine.
  • 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.
  • an 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 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 comprises phosphodiester linkages between nucleotides that do not comprise a phosphorothioate linkage.
  • 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 internucleotide linkage backbone of methylene morpholine rings linked through phosphorodiamidate groups.
  • PMO morpholino oligomer
  • Base Modifications In some embodiments, an RNAi oligonucleotide herein comprises one or more modified nucleobases.
  • modified nucleobases are linked at the 1 ⁇ position of a nucleotide sugar moiety.
  • 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 T m than a duplex formed with the nucleic acid comprising the mismatched base.
  • universal-binding nucleotides include, but are not limited to, inosine, 1- ⁇ -D-ribofuranosyl-5-nitroindole and/or 1- ⁇ -D-ribofuranosyl-3-nitropyrrole (see, US Patent Application Publication No.2007/0254362; Van Aerschot et al.
  • 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. In some embodiments, 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.
  • the oligonucleotide has up to seven Tm- increasing nucleotides in the sense strand. In some embodiments, the oligonucleotide has up to FH12501620.1 Attorney Docket: DCY-13025 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. In some embodiments, an oligonucleotide comprising a stem-loop comprises a Tm- increasing nucleotide in the stem.
  • 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 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.
  • the oligonucleotide-ligand conjugates of the disclosure comprise are bicyclic nucleotides.
  • Formula (AI), (AII), (BI), (BII), (CI), or (CII) comprise are bicyclic nucleotides.
  • FH12501620.1 Attorney Docket: DCY-13025 the monocyclic sugar moiety shown in Formula (AI), (AII), (BI), (BII), (CI), or (CII) may be replaced by a bicyclic sugar as discussed below.
  • the T m -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.
  • the bridge contains 3 atoms.
  • the bridge contains 4 atoms.
  • the bridge contains 5 atoms.
  • the bridge contains 6 atoms.
  • the bridge contains 7 atoms.
  • the bridge contains 8 atoms.
  • 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: FH12501620.1 Attorney Docket: DCY-13025 wherein B is a nucleobase; wherein G is H, OH, NH 2 , C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 1 -C 6 alkyl, substituted C 2 -C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thio; wherein X is O, S, or NR 1 , wherein R 1 is H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, benzene or pyrene; and wherein Wa and Wb are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group
  • the bicyclic nucleotide has the structure of Formula II: Formula II wherein B is a nucleobase; wherein Q 1 is CH 2 or O; wherein X is CH 2 , O, S, or NR 1 , wherein R 1 is H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, benzene or pyrene; wherein if Q1 is O, X is CH2; wherein if Q1 is CH2, X is CH2, O, S, or or
  • Q2 is O and X is NR1. In some embodiments of Formula III, Q2 is O and X is NR1, wherein R1 is C1-C6 alkyl. In some embodiments of Formula III, Q2 is O and X is NR1 and R1 is H or CH3. In some embodiments of Formula III, Q 2 is O and X is NR 1 and R 1 is CH 3 : Formula IIIa In some embodiments of Formula III, Q 2 is NR 1 and X is O. In some embodiments of Formula III, Q 2 is NR 1 , wherein R 1 is C 1 -C 6 alkyl and X is O.
  • the bicyclic nucleotide has the structure of Formula IV: FH12501620.1 Attorney Docket: DCY-13025 Formula IV wherein B is a nucleobase; wherein P 1 and P 3 are CH 2 , P 2 is CH 2 or O and P 4 is O; and wherein W a and W b 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 W a or Wb is an internucleotide linking group attaching the nucleotide represented by Formula IV to an oligonucleotide.
  • the bicyclic nucleotide has the structure of Formula Va or Vb: FH12501620.1 Attorney Docket: DCY-13025 wherein B is a nucleobase; wherein r1, r2, r3, and r4 are each independently H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl; substituted C2-C12 alkynyl; C1-C12 alkoxy; substituted C1-C12 alkoxy, OT1, ST1, SOT1, SO2T1, NT1T2, N3, CN, C( ⁇ O)OT 1 , C( ⁇ O)NT 1 T 2, C( ⁇ O)
  • 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, C 1 -C 12 alkyl, or a protecting group, including, for example, 4 ⁇ -CH 2 -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 ⁇ -CH2-2 ⁇ ; 4 ⁇ -(CH2)2-2 ⁇ ; 4 ⁇ -(CH2)3-2 ⁇ ; 4 ⁇ -(CH2)-O-2 ⁇ (also known as LNA); 4 ⁇ -(CH2)-S-2 ⁇ ; 4 ⁇ -(CH2)2-O-2 ⁇ (also known as ENA); 4 ⁇ -CH(CH3)-O-2 ⁇ FH12501620.1
  • Patent Publication No.2004/0171570 which is hereby incorporated by reference in its entirety
  • f) 4 ⁇ -CH2-C(H)(CH3)-2 ⁇ and analogs thereof as described in Chattopadhyaya et al., J. O RG . C HEM ., 2009, 74, 118-34, which is hereby incorporated by reference in its entirety
  • 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
  • R 2 is H or CH 3
  • W a and W b are each independently, H, OH, a hydroxyl protecting group, a phosphorous moiety, or an internucleotide linking group attaching the bicyclic nucleotide to another nucleotide or to an oligonucleotide and wherein at least one of Wa or Wb is an internucleotide linking group attaching the bicyclic nucleotide to an oligonucleotide.
  • R2 is CH3, as follows (also known as BNA NC [NMe]): .
  • 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 ⁇ -L configuration.
  • the bicyclic sugar moiety or nucleotide is in the ⁇ -D configuration.
  • the bicyclic sugar moiety or nucleotide comprises a 2 ⁇ O,4 ⁇ -C-methylene bridge (2 ⁇ -O-CH 2 -4 ⁇ ) in the ⁇ -L configuration ( ⁇ -L LNA).
  • the bicyclic sugar moiety or nucleotide is in the R configuration.
  • the bicyclic sugar FH12501620.1 Attorney Docket: DCY-13025 moiety or nucleotide is in the S configuration.
  • the bicyclic sugar moiety or nucleotide comprises a 4 ⁇ -CH(CH 3 )-O-2 ⁇ bridge (i.e., cEt) in the S- configuration.
  • Tricyclic Nucleotides In some embodiments, the Tm-increasing nucleotide is a tricyclic nucleotide.
  • the tricyclic nucleotide is a tricyclo nucleotide (also called tricyclo 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.
  • 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.
  • Tm-increasing nucleotides In addition to bicyclic and tricyclic nucleotides, other T m -increasing nucleotides can be used in the RNAi oligonucleotides described herein.
  • the T m -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.
  • 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 T m -increasing nucleotide is not modified at the 2 ⁇ -carbon of the sugar moiety with a 2 ⁇ -F or a 2 ⁇ -OMe.
  • the T m -increasing nucleotide is a bicyclic nucleotide.
  • the T m -increasing nucleotide is a tricyclic nucleotide.
  • the Tm-increasing nucleotide a G-clamp, guanidine G-clamp or analogue thereof.
  • the Tm-increasing nucleotide is a hexitol nucleotide. In some embodiments, the Tm-increasing nucleotide is a bicyclic or tricyclic nucleotide. In some embodiments, 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. In some embodiments, the Tm-increasing nucleotide increases the Tm of the nucleic acid inhibitor molecule by at least 2 °C per incorporation. In some embodiments, the T m -increasing nucleotide increases the T m of nucleic acid inhibitor molecule by at least 3 °C per incorporation.
  • the T m -increasing nucleotide increases the T m of nucleic acid inhibitor molecule by at least 4 °C per incorporation. In some embodiments, the T m -increasing nucleotide increases the Tm of nucleic acid inhibitor molecule by at least 5 °C per incorporation.
  • mRNA Target Sequences In some embodiments, the RNAi oligonucleotide is targeted to a target sequence comprising a target mRNA. In some embodiments, the RNAi oligonucleotide is targeted to a target sequence within a target mRNA.
  • the RNAi oligonucleotide, or a portion, fragment, or strand thereof binds or anneals to a target sequence comprising a target mRNA, thereby reducing target gene expression.
  • the 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 an RNAi oligonucleotide targeted to a specific target sequence correlates with the potency of the RNAi oligonucleotide. In some embodiments, the amount or extent of reduction of target gene expression by an 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 RNAi oligonucleotide.
  • FH12501620.1 Attorney Docket: DCY-13025
  • 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.
  • a sense strand of an 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 hepatic tissue, adipose tissue, cardiac tissue, adrenal tissue, or skeletal muscle tissue. In some embodiments, the target mRNA is expressed in extrahepatic tissue.
  • the target mRNA is expressed in cardiac tissue, adipose tissue, adrenal tissue, the central nervous system, or skeletal muscle tissue. In some embodiments, the target mRNA is expressed in cardiac tissue. In some embodiments, the target mRNA is expressed in adipose tissue. In some embodiments, the target mRNA is expressed in gonadal white adipose tissue. In some embodiments, the target mRNA is expressed in subcutaneous white 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 the central nervous system.
  • RNAi oligonucleotide it is desirable to target the oligonucleotides of the disclosure (e.g., RNAi oligonucleotide) to one or more cells or tissues of extrahepatic tissue. In some embodiments, it is desirable to target the oligonucleotides of the disclosure (e.g., RNAi oligonucleotide) to one or more cells or tissues of the liver. In some embodiments, it is desirable to target the oligonucleotides of the disclosure (e.g., RNAi oligonucleotide) to one or more cells or tissues of extrahepatic tissue.
  • RNAi oligonucleotide it is desirable to target the oligonucleotides of the disclosure (e.g., RNAi oligonucleotide) to one or more cells or tissues of the heart. In some embodiments, it is desirable to target the oligonucleotides of the disclosure (e.g., RNAi oligonucleotide) to one or more cells or tissues of the skeletal muscle. In some embodiments, it is desirable to target the oligonucleotides of the disclosure (e.g., RNAi 111 FH12501620.1 Attorney Docket: DCY-13025 oligonucleotide) to one or more cells or tissues of the adrenal gland.
  • RNAi oligonucleotide e.g., RNAi oligonucleotide
  • RNAi oligonucleotide e.g., RNAi oligonucleotide
  • a strategy can help to avoid undesirable effects in other organs or avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit from the oligonucleotide.
  • an 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 extrahepatic tissue).
  • an 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). In some embodiments, an RNAi oligonucleotide herein does not have a GalNAc conjugated thereto. In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an RNAi oligonucleotide disclosed herein are each conjugated to a separate targeting ligand. In some embodiments, 1 nucleotide of an RNAi oligonucleotide herein is conjugated to a separate targeting ligand.
  • nucleotide of an RNAi oligonucleotide herein is conjugated to a separate targeting ligand.
  • 2 to 4 nucleotides of an RNAi oligonucleotide herein are each conjugated to a separate targeting ligand. In some embodiments, 2 nucleotides of an RNAi oligonucleotide herein are each conjugated to a separate targeting ligand. In some embodiments, the 5’ terminal nucleotide of the sense strand and the 3’ terminal nucleotide of the sense strand are conjugated to a targeting ligand described herein. In some embodiments, the 5’ terminal nucleotide of the sense strand and the 3’ terminal nucleotide of the sense strand are conjugated to the same targeting ligand.
  • the 5’ terminal nucleotide of the sense strand and the 3’ terminal nucleotide of the sense strand are conjugated to different targeting ligands. In some embodiments, nucleotides at position 1 and position 2 of the sense strand are conjugated to the same targeting ligand. In some embodiments, nucleotides at position 1 and position 2 of the sense strand are conjugated to different targeting ligands.
  • 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 RNAi oligonucleotide resembles a toothbrush.
  • an 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.
  • an 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.
  • 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 Intl. Patent Application Publication No. WO 2016/100401.
  • the linker is a labile linker. However, in other embodiments, the linker is stable.
  • 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 Intl. Patent Application Publication No. WO 2016/100401.
  • the linker is a labile linker.
  • the linker is a stable linker.
  • the linker is In some embodiments, the linker is In some embodiments, a duplex extension (e.g., of up to 3, 4, 5 or 6 bp in length) is provided between a targeting ligand and an RNAi oligonucleotide.
  • Ligand Conjugates FH12501620.1 Attorney Docket: DCY-13025 any of the ligands described herein are conjugated to a nucleotide of the sense strand of the oligonucleotide. In some embodiments, a ligand is conjugated to a terminal position of the oligonucleotide (e.g., as shown in Formula (BI), (BII), (CI) or (CII)).
  • the ligand is conjugated to the 5’ terminal nucleotide of the sense strand (e.g., as shown in Formula (BI) or (BII)). In some embodiments, the ligand is conjugated to the 3’ terminal nucleotide of the sense strand (e.g., as shown in Formula (CI) or (CII)). In some embodiments, the ligand is conjugated to an internal nucleotide on the sense strand (e.g., as shown in Formula (AI)-(AV)). An internal position is any nucleotide position other than the two terminal positions from each end of the sense strand. In some embodiments, the ligand is conjugated to one or more internal positions of the sense strand.
  • the ligand 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 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37 or position 38 of a sense strand.
  • the ligand is conjugated to position 1 of the sense strand.
  • the ligand is conjugated to position 28 of the sense strand.
  • two ligands are conjugated to the sense strand of the oligonucleotide.
  • the two ligands conjugated to the sense strand of the oligonucleotide are the same ligand. In some embodiments, the two ligands conjugated to the sense strand of the oligonucleotide are different ligands. In some embodiments, the two ligands conjugated to the sense strand are conjugated to position 1 and to a nucleotide in the stem loop. In some embodiments, the two ligands conjugated the sense strand are conjugated to position 1 and position and position 28. In some embodiments, the two ligands conjugated to the sense strand are conjugated at position 2 and position 29.
  • an RNAi oligonucleotide described herein comprises at least one nucleotide conjugated with one or more ligands.
  • the one or more ligands are conjugated to the same nucleotide.
  • the one or more ligands are conjugated to different nucleotides.
  • 1, 2, 3, 4, 5, 6, or more ligands are conjugated to the oligonucleotide.
  • one or more ligands are conjugated to an adenine nucleotide.
  • the hydrocarbon chain comprises at least 6 (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or at least 26) carbon atoms.
  • the hydrocarbon chain comprises from 6-26 carbon atoms (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26).
  • 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 5’ end of the antisense strand is a blunt end.
  • the 3’ end of the antisense strand comprises an overhang.
  • the 5’ end of the antisense strand comprises an overhang.
  • the 5’ and 3’ ends of the antisense strand each comprise an overhang.
  • the RNAi oligonucleotide comprises one or more 2’ modifications. In some embodiments, the 2’ modifications are selected from 2’-fluoro and 2’- methyl.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-(CO2H)n groups, or a charged form thereof, wherein L is a linker, Y is alkylene, alkenylene, or alkynylene, and n is 1-6, wherein when Y is alkylene, L comprises -O(CH2)a(OCH2CH2)bNH-, wherein a is 1- 6 and b is 0-5, and wherein the ligand is conjugated to a nucleo
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length, a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra- hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more C6-24 alkylene-(CO2H)n, or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein n is 1-6, wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 36 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is FH12501620.1 Attorney Docket: DCY-13025 , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length, a sense strand of 36 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and FH12501620.1 Attorney Docket: DCY-13025 (ii) one or more ligands, wherein the ligand is selected from , wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-(CO2H)n groups, or a charged form thereof, wherein L is a linker, Y is alkylene, alkenylene, or alkynylene, and n is 1-6, wherein when Y is alkylene, L comprises -O(CH2)a(OCH2CH2)bNH-, wherein a is 1- 6 and b is 0-5, and wherein the ligand is conjugated to a nucleotide of the
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and FH12501620.1 Attorney Docket: DCY-13025 (ii) one or more ligands, each ligand comprising one or more C6-24 alkylene-(CO2H)n, or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein n is 1-6, wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is ,wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is FH12501620.1 Attorney Docket: DCY-13025 , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is ,wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is selected from FH12501620.1 Attorney Docket: DCY-13025 , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 22 nucleotides in length, a sense strand of 37 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is selected from FH12501620.1 Attorney Docket: DCY-13025 ,wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 20 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-(CO2H)n groups, or a charged form thereof, wherein L is a linker, Y is alkylene, alkenylene, or alkynylene, and n is 1-6, wherein when Y is alkylene, L comprises -O(CH 2 ) a (OCH 2 CH 2 ) b NH-, wherein a is 1- 6 and b is 0-5, and wherein the ligand is conjugated to a
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 20 nucleotides in length, a sense strand of 36 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and FH12501620.1 Attorney Docket: DCY-13025 (ii) one or more ligands, each ligand comprising one or more C6-24 alkylene-(CO2H)n, or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein n is 1-6, wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 20 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 20 nucleotides in length, a sense strand of 36 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is ,wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 20 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is FH12501620.1 Attorney Docket: DCY-13025 , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 20 nucleotides in length, a sense strand of 36 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, wherein the ligand is ,wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises: (i) an antisense strand of 22 nucleotides in length, a sense strand of 20 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and FH12501620.1 Attorney Docket: DCY-13025 (ii) one or more ligands, wherein the ligand is selected from , and wherein the ligand is conjugated to a nucleotide of the sense strand.
  • an RNAi oligonucleotide comprises: (i) a double-stranded oligonucleotide comprising an antisense strand of 20 nucleotides in length, a sense strand of 36 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extra-hepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and FH12501620.1 Attorney Docket: DCY-13025 (ii) one or more ligands, wherein the ligand is selected from ,wherein knock down of the target mRNA in the extra-hepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 20 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one oligonucleotide-ligand conjugate, and wherein the oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 36 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one oligonucleotide-ligand conjugate, and wherein the oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 37 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one FH12501620.1 Attorney Docket: DCY-13025 oligonucleotide-ligand conjugate, and wherein the oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 20 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one oligonucleotide-ligand conjugate, wherein the oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), and wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 20 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one oligonucleotide-ligand conjugate, wherein the oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), and wherein the nucleobase (B) is the nucleobase at position 20 of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 36 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one oligonucleotide-ligand conjugate, wherein the oligonucleotide- ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), and wherein the nucleobase (B) is the nucleobase at position 28 of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 37 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one oligonucleotide-ligand conjugate, wherein the oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), and wherein the nucleobase (B) is the nucleobase at position 29 of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 36 nucleotides in length, and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises at least one oligonucleotide-ligand conjugate, wherein the oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), and wherein the nucleobase (B) is the nucleobase in the tetraloop of the sense strand.
  • an RNAi oligonucleotide comprises an oligonucleotide-ligand conjugate of any one of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII). In some embodiments, an RNAi oligonucleotide comprises at least one oligonucleotide-ligand conjugate of any of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprises at least two oligonucleotide-ligand FH12501620.1 Attorney Docket: DCY-13025 conjugates selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII).
  • an RNAi oligonucleotide comprises at least two oligonucleotide-ligand conjugates selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the at least two oligonucleotide-ligand conjugates are selected from the same Formula.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of any one of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 28 of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises an oligonucleotide-ligand conjugate of any one of Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 29 of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises a first and a second oligonucleotide-ligand conjugate, wherein: FH12501620.1 Attorney Docket: DCY-13025
  • the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand
  • the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), or (AV), wherein the nucleobase (B) is the nucleobase at position 2 of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises a first and a second oligonucleotide-ligand conjugate, wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), or (AV), wherein the nucleobase (B) is the nucleobase at position 20 of the sense strand.
  • the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI),
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises a first and a second oligonucleotide-ligand conjugate, wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is a nucleobase in the tetraloop of the sense strand.
  • the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (A
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises a first and a second oligonucleotide-ligand conjugate, wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 28 of the sense strand.
  • the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises a first and a second oligonucleotide-ligand conjugate, wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), or (AV), wherein the nucleobase (B) is the nucleobase at position 2 of the sense strand.
  • the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI),
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand comprises a first and a second oligonucleotide-ligand conjugate, wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 20 of the sense strand.
  • the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 20 nucleotides in length and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises a first and a second oligonucleotide- ligand conjugate, and wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 20 of the sense strand
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 36 nucleotides in length and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises a first and a second oligonucleotide- ligand conjugate, and wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), or (AV), wherein the nucleobase (B) is a nucleobase in the tetraloop of the sense strand.
  • an RNAi oligonucleotide comprises a sense and antisense strand, wherein the sense strand is 37 nucleotides in length and the antisense strand is 22 nucleotides in length, wherein the sense strand comprises a first and a second oligonucleotide- ligand conjugate, and wherein: (i) the first oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), (AV), (BI), (BII), (CI), or (CII), wherein the nucleobase (B) is the nucleobase at position 1 of the sense strand; and (ii) the second oligonucleotide-ligand conjugate is selected from Formulas (AI), (AII), (AIII), (AIV), or (AV), wherein the nucleobase (B) is the nucleobase at position 29 of the
  • nucleic acids and analogues thereof comprising an oligonucleotide-ligand 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 some 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 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 internucleotide 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 nucle
  • an oligonucleotide is prepared comprising 1-3 nucleic acid or analogues thereof comprising hydrocarbon chain bearing one or more carboxyl groups on a tetraloop.
  • Scheme A where a particular protecting group, leaving group, or transformation condition is depicted, one of ordinary skill in the art will appreciate that other protecting groups, leaving groups, and transformation conditions are also suitable and are contemplated.
  • nucleic acids, and analogues thereof of the present disclosure are generally prepared according to Scheme A, Scheme A1 and Scheme B set forth below: FH12501620.1 Attorney Docket: DCY-13025 Scheme A: Synthesis of Ligand Conjugated Oligonucleotides of the Disclosure Scheme A1: Synthesis of Conjugated Oligonucleotides of the Disclosure FH12501620.1 Attorney Docket: DCY-13025 FH12501620.1 Attorney Docket: DCY-13025 As depicted in Scheme A and Scheme A1 above, a nucleic acid or analogue thereof of formula I-1 is conjugated with one or more ligand/lipophilic compound to form a compound of formula I or Ia comprising one more ligand conjugates.
  • conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula I-1 or I-1a and one or more hydrocarbon bearing carboxylic acid in series or in parallel by known techniques in the art.
  • Nucleic acid or analogue thereof of formula I or Ia can then be deprotected to form a compound of formula I-2 or I-2a and protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula I-3 or I-3a.
  • a suitable hydroxyl protecting group e.g., DMTr
  • nucleic acid-ligand conjugates of formula I-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 I-4 or I-4a comprising one or more hydrocarbon with carboxylic acid.
  • a nucleic acid- ligand conjugates of formula I-3 or I-3a can react with a P(III) forming reagent (e.g., 2-cyanoethyl N,N-di- isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula I-5 or I-5a comprising a P(III) group.
  • a P(III) forming reagent e.g., 2-cyanoethyl N,N-di- isopropylchlorophosphoramidite
  • a nucleic acid-ligand conjugate or analogue thereof of formula I-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 I-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 conjugate nucleotide units represented by a compound of formula II-1 or II-Ia.
  • Each of B, L, R 1 , R 2 , and Z is as defined above and in Formula (AI), (BI), or (CI) or as described herein.
  • X as provided in formula I-1, is a reactive functional group or leaving group.
  • PG 1 and PG 2 comprise silyl ethers or cyclic silylene derivatives that can be removed under acidic conditions or with fluoride anion.
  • PG 1 and PG 2 are taken together with their intervening atoms to form a cyclic diol protecting group, such as a cyclic acetal or ketal.
  • PG 3 used for protection of the 5’-hydroxyl group includes an acid labile protecting group such as trityl, 4-methyoxytrityl, 4,4’-dimethyoxytrityl, 4,4’,4’’-trimethyoxytrityl, 9-phenyl-xanthen- 9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
  • E is 2-cyanoethyl when 2-cyanoethyl N,N-diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate is used as a P(III) forming reagent.
  • X 3 is O-, -S-, or a covalent bond.
  • R 3 is hydrogen or a suitable protecting group.
  • n is 1, 2, or 3.
  • FH12501620.1 Attorney Docket: DCY-13025
  • Scheme B Post-Synthetic Conjugation of Oligonucleotides of the Disclosure
  • a nucleic acid or analogue thereof of formula I-1 can be deprotected to form a compound of formula I-6, protected with a suitable hydroxyl protecting group (e.g., DMTr) to form a compound of formula I-7, and reacted with a P(III) forming reagent (e.g., 2-cyanoethyl N,N-di-isopropylchlorophosphoramidite) to form a nucleic acid or analogue thereof of formula I-8 comprising a P(III) group.
  • a suitable hydroxyl protecting group e.g., DMTr
  • P(III) forming reagent
  • a nucleic acid or analogue thereof of formula I-8 is subjected to oligomerization forming conditions preformed using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula I-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.
  • conjugation is performed through an esterification or amidation reaction between a nucleic acid or analogue thereof of formula II-2 and one or more hydrocarbon bearing carboxylic 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 3 , R 1 , R 2 , R 3 , X 3 , and Z is as defined above.
  • nucleic acids, and analogues thereof of the present disclosure FH12501620.1 Attorney Docket: DCY-13025 are prepared according to Scheme C and Scheme D set forth below:
  • Scheme C Synthesis of Conjugated Oligonucleotides of the Disclosure
  • a nucleic acid or analogue thereof of formula C1 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 hydrocarbon chain bearing one or more carboxyl groups FH12501620.1 Attorney Docket: DCY-13025 of the disclosure.
  • a ligand adamantyl or lipophilic compound (e.g., a fatty acid)
  • amide forming conditions e.g., HATU, DIPEA
  • 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.
  • a suitable hydroxyl protecting group e.g., DMTr
  • 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 C10 comprising a ligand conjugate 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 N,N-di-isopropylchlorophosphoramidite) to form a nucleic acid-ligand conjugate or analogue thereof of formula C11 comprising a P(III) group.
  • a nucleic acid-ligand conjugate or analogue thereof of formula C11 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 C11 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 conjugate nucleotide units represented by a compound of formula II-b-3.
  • Each of B, E, PG 1 , PG 2 , PG 3 , R 1 , R 2 , R 3 , R 4 , a, b, X 1 , X 2 , X 3 , Y, and Z is as defined above.
  • a nucleic acid or analogue thereof of formula C5 can be selectively deprotected to form a compound of formula D1, 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 N,N-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., hydrocarbon chain moiety) to form a compound of formula C7 (e.g., hydrocarbon chain moeity) under appropriate amide forming conditions (e.g., HATU, DIPEA), to form an oligonucleotide of formula II-b-3 comprising a ligand (e.g., a fatty acid) conjugate of the disclosure.
  • a hydrophobic ligand e.g., hydrocarbon chain moiety
  • C7 e.g., hydrocarbon chain moeity
  • appropriate amide forming conditions e.g., HATU, DIPEA
  • nucleic acid or analogues thereof of the disclosure such as aliphatic groups, alcohols, FH12501620.1 Attorney Docket: DCY-13025 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 conjugate, said conjugate unit represent by formula II-a-1: II-a-1 or a pharmaceutically acceptable salt thereof, comprising the steps of: (a) providing a nucleic acid or analogue thereof of formula I-5a: or salt thereof, and (b) oligomerizing said compound of formula I-5a to form a compound of formula II-1a, wherein each of B, E, L, LC, n, PG 3 , R 1 , R 2 , R 3 , X 3 , and Z is as defined above and described herein.
  • 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 II-1a FH12501620.1 Attorney Docket: DCY-13025 comprising a conjugate of the disclosure.
  • the present disclosure provides a method for preparing an oligonucleotide comprising one or more conjugate, further comprising preparing a nucleic acid or analogue thereof of formula I-5a: I-5a or a salt thereof, comprising the steps of: (a) providing a nucleic acid or analogue thereof of formula Ia: or salt thereof, (b) deprotecting said nucleic acid or analogue thereof of formula Ia to form a compound of formula I-2a: or salt thereof, (c) protecting said nucleic acid or analogue thereof of formula I-2 to form a compound of formula I-3a: I-3a or salt thereof, and FH12501620.1 Attorney Docket: DCY-13025 (d) treating said 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 3 , R 1
  • PG 1 and PG 2 of a compound of formula Ia 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-N-butylammonium fluoride, and the like.
  • step (c) above a compound of formula I-2a is protected with a suitable hydroxyl protecting group.
  • the protecting group PG 3 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-methyoxytrityl, 4,4’-dimethyoxytrityl, 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, dichloroacetic acid or trichloroacetic acid.
  • a compound of formula I-3a is treated with a P(III) forming reagent to afford a compound of formula I-5a.
  • 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. In some embodiments, the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
  • suitable bases are well known in the art and include organic and inorganic bases.
  • the base is a tertiary amine such as triethylamine or diisopropylethylamine.
  • step (d) above is preformed using N,N-dimethylphosphoramic dichloride as a P(V) forming reagent.
  • 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-Cl, 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-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • the present disclosure provides a method for preparing an oligonucleotide comprising one or more conjugates, said conjugate unit represent by formula II-1: FH12501620.1 Attorney Docket: DCY-13025 II-1 or a pharmaceutically acceptable salt thereof, comprising the steps of: (a) providing an oligonucleotide of formula II-2: II-2 or salt thereof, and, (b) conjugating one or more lipophilic compounds to an oligonucleotide of formula II-2 to form an oligonucleotide of formula II-1 comprising one or more conjugates.
  • an oligonucleotide of formula II-2 is conjugated with one or more lipophilic compounds to form an oligonucleotide of formula II-1 comprising one more 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-1 comprising one more 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-Cl, 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-Cl, DEPBT, T3P, TATU, TBTU, TNTU, TOTU, TPTU, TSTU, or TDBTU.
  • the present disclosure provides a method for preparing an oligonucleotide comprising a unit represent by formula II-2: FH12501620.1 Attorney Docket: DCY-13025 or a pharmaceutically acceptable salt thereof, comprising the steps of: (a) providing a nucleic acid or analogue thereof of formula I-8: I-8 or salt thereof, and (b) oligomerizing said compound of formula I-8 to form a compound of formula II-2.
  • oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
  • the present disclosure provides a method for preparing a nucleic acid or analogue thereof comprising one or more conjugates, further comprising preparing a nucleic acid or analogue thereof of formula I-8: I-8 or a salt thereof, comprising the steps of: (a) providing a nucleic acid or analogue thereof of formula I-1: I-1 or salt thereof, (b) deprotecting said nucleic acid or analogue thereof of formula I-1 to form a compound of formula I-6: FH12501620.1 Attorney Docket: DCY-13025 I-6 or salt thereof, (c) protecting said nucleic acid or analogue thereof of formula I-6 to form a compound of formula I-7: I-7 or salt thereof, and (d) treating said nucleic acid or analogue thereof of formula I-7 with a P(III) forming reagent to form a nucleic acid or analogue thereof of formula I-8,
  • PG 1 and PG 2 of a compound of formula I-1 comprise sily
  • reagents providing fluoride anion for the removal of silicon-based protecting groups include hydrofluoric acid, hydrogen fluoride pyridine, triethylamine trihydrofluoride, tetra-N-butylammonium fluoride, and the like.
  • step (c) above a compound of formula I-6 is protected with a suitable hydroxyl protecting group.
  • the protecting group PG 3 used for protection of the 5’-hydroxyl group of a compound of formula I-6 includes an acid labile protecting group such as trityl, 4-methyoxytrityl, 4,4’-dimethyoxytrityl, 4,4’,4’’-trimethyoxytrityl, 9-phenyl- xanthen-9-yl, 9-(p-tolyl)-xanthen-9-yl, pixyl, 2,7-dimethylpixyl, and the like.
  • the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite or 2-cyanoethyl phosphorodichloridate.
  • the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
  • the base is a tertiary amine such as triethylamine or diisopropylethylamine.
  • step (d) above is preformed using N,N-dimethylphosphoramic dichloride as a P(V) forming reagent.
  • the present disclosure provides a method for preparing an oligonucleotide-ligand conjugate comprising one or more Y-COOH groups, said conjugate unit represented by formula II-b-3: II-b-3 or a pharmaceutically acceptable salt thereof, comprising the steps of: (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula C11: or salt thereof, and (b) oligomerizing said compound of formula C11 to form a compound of formula II-b-3, In step (b) above, oligomerizing refers to preforming oligomerization forming conditions using known and commonly applied processes to prepare oligonucleotides in the art.
  • the compound of formula C11 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 C11: or a salt thereof, comprising the steps of: (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula I-b: I-b or salt thereof, (b) deprotecting said nucleic acid-ligand conjugate or analogue thereof of formula I-b to form a compound of formula C8: C8 or salt thereof, (c) protecting said nucleic acid-ligand conjugate or analogue thereof of formula C8 to form a compound of formula C9: or salt thereof, and (d) treating said nucleic acid-ligand conjugate or analogue thereof of formula C9 with a FH12501620.1 Attorney Docket: DCY-13025 P(III) forming reagent to form
  • 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-N- butylammonium fluoride, and the like.
  • step (c) above 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-methyoxytrityl, 4,4’-dimethyoxytrityl, 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, dichloroacetic acid or trichloroacetic acid.
  • a compound of formula C9 is treated with a P(III) forming reagent to afford a compound of formula C11.
  • 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. In some embodiments, the P(III) forming reagent is 2-cyanoethyl N,N- diisopropylchlorophosphoramidite.
  • suitable bases are well known in the art and include organic and inorganic bases.
  • the base is a tertiary amine such as triethylamine or diisopropylethylamine.
  • step (d) above is preformed using N,N-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: FH12501620.1 Attorney Docket: DCY-13025 or a salt thereof, comprising the steps of: (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula C6: or salt thereof, and, (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.
  • 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-Cl, 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: FH12501620.1 Attorney Docket: DCY-13025 C6 or a salt thereof, comprising the steps of: (a) providing a nucleic acid or analogue thereof of formula C1: C1 or salt thereof, and, (b) protecting said nucleic acid or analogue thereof of formula C1 to form a compound of formula C2: C2 or salt thereof, (c) alkylating said nucleic acid or analogue thereof of formula C2 to form a compound of formula C3: C3 or salt thereof, (d) substituting said nucleic acid or analogue thereof of formula C3 with a compound of formula C4: C4 or salt thereof, to form a compound of formula C5: FH12501620.1
  • 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.
  • a cyclic diol protecting group such as a cyclic acetal or ketal.
  • 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).
  • cAMP cyclic adenosine monophosphate
  • the cyclic diol protection group is 1,1,3,3- tetraisopropylidisiloxanylidene prepared from the reaction of a diol of formula C1 and 1,3- dichloro-1,1,3,3-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.
  • the 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.
  • the mild oxidation reagent includes a mixture of elemental iodine and hydrogen peroxide, urea hydrogen peroxide complex, silver nitrate/silver sulfate, sodium bromate, ammonium peroxodisulfate, tetrabutylammonium peroxydisulfate, Oxone®, Chloramine T, Selectfluor®, Selectfluor® II, sodium hypochlorite, or potassium iodate/sodium periodiate.
  • the mild oxidizing agent includes N-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide, 1,3-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 4 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 FH12501620.1 Attorney Docket: DCY-13025 a nucleic acid-ligand conjugate or analogue thereof of formula C6 or a salt thereof.
  • PG 4 and/or R 4 comprise carbamate derivatives that can be removed under acidic or basic conditions.
  • the protecting groups e.g., both PG 4 and R 4 or either of PG 4 or R 4 independently
  • the protecting groups of a nucleic acid-ligand conjugate or analogue thereof of formula C5 are removed by acid hydrolysis. It will be appreciated that 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. For example, when 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.
  • a wide variety of 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 4 and R 4 or either of PG 4 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).
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-ene
  • 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: II-b-3 or a pharmaceutically acceptable salt thereof, comprising the steps of: (a) providing an oligonucleotide of formula D5: FH12501620.1 Attorney Docket: DCY-13025 or salt thereof, and, (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.
  • 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-Cl, 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: (a) providing a nucleic acid-ligand conjugate or analogue thereof of formula D4: D4 or salt thereof, and (b) deprotecting said compound of formula D4 to form a compound of formula D5.
  • step (b) above removal of PG 4 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 FH12501620.1 Attorney Docket: DCY-13025 or a salt thereof.
  • PG 4 and/or R 4 comprise carbamate derivatives that can be removed under acidic or basic conditions.
  • the protecting groups (e.g., both PG 4 and R 4 or either of PG 4 or R 4 independently) of an oligonucleotide-ligand conjugate of formula D4 are removed by acid hydrolysis.
  • the protecting groups e.g., both PG 4 and R 4 or either of PG 4 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).
  • 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 D4: or a pharmaceutically acceptable salt thereof, comprising the steps of: (a) providing a nucleic acid or analogue thereof of formula D3: FH12501620.1 Attorney Docket: DCY-13025 or salt thereof, and (b) oligomerizing said compound of formula D3 to form a compound of formula D4, In step (b) above, 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.
  • oligonucleotides e.g., RNAi oligonucleotides
  • a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation.
  • compositions comprising oligonucleotides (e.g., RNAi oligonucleotide) reduce the expression of a target mRNA (e.g., a target mRNA expressed in adipose tissue).
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, FH12501620.1 Attorney Docket: DCY-13025 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.
  • 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.
  • Another key factor is the stereochemical effect that arises in oligomers having P-chiral centers. In general, an oligomer with a length of n nucleosides will constitute a mixture of chirality in successive non-stereospecific chain synthesis.
  • the effect of delivery of an 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).
  • reduction in expression of a target gene in liver tissue 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 the reduction in expression of the target gene in extrahepatic tissue.
  • differences in target mRNA expression between cell types or tissue types is measured using methods known in the art.
  • differences in target mRNA expression between cell types or tissue types measures the reduction of the target mRNA in a first cell/tissue type compared to the reduction of target mRNA in a second cell/tissue type.
  • differences in target mRNA expression between cell types or tissue types is measured using polymerase chain reaction methods (e.g., RT-PCR) comparing relative expression between different tissue or cell types.
  • differences in target mRNA expression between cell types or tissue types is measured using Northern blot analysis, in situ hybridization, RT-PCR, RNA sequencing, or other methods known in the art.
  • a relative amount of target mRNA expression is compared between cell or tissue types.
  • an absolute amount of target mRNA expression is compared between cell or tissue types. Reducing Target Gene Expression in Cardiac Tissue
  • expression of a target gene is reduced in a region of cardiac tissue.
  • expression of a target gene in the cardiac tissue 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 other control tissue.
  • reduction in expression of a target gene in liver tissue 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 the reduction in expression of the target gene in cardiac tissue.
  • Reducing Target Gene Expression in Skeletal Muscle In some embodiments, expression of a target gene is reduced in skeletal muscle.
  • expression of a target gene in the skeletal muscle 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 other control tissue.
  • expression of a target gene in the skeletal muscle 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 liver tissue.
  • contacting or delivering an RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene in skeletal muscle.
  • the reduction in expression of a target gene in skeletal muscle is relative to an amount or level of target gene expression in liver tissue contacted with the RNAi oligonucleotide.
  • reduction in expression of a target gene in the skeletal muscle of a subject 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 30%, at least 35%, at least 40%, FH12501620.1 Attorney Docket: DCY-13025 at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to reduction in expression of the target gene in liver tissue.
  • reduction in expression of a target gene in liver tissue 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 the reduction in expression of the target gene in skeletal muscle.
  • Reducing Target Gene Expression in Adrenal Gland In some embodiments, expression of a target gene is reduced in a region of the adrenal gland.
  • expression of a target gene in the adrenal gland 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 other control tissue.
  • expression of a target gene in the adrenal gland 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 liver tissue.
  • contacting or delivering an RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene in adrenal gland.
  • the reduction in expression of a target gene in adrenal gland is relative to an amount or level of target gene expression in liver tissue contacted with the RNAi oligonucleotide.
  • reduction in expression of a target gene in the adrenal gland of a subject 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 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to reduction in expression of the target gene in liver tissue.
  • reduction in expression of a target gene in liver tissue 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 FH12501620.1 Attorney Docket: DCY-13025 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to the reduction in expression of the target gene in adrenal gland.
  • Reducing Target Gene Expression in Adipose Tissue In some embodiments, expression of a target gene is reduced in adipose tissue.
  • expression of a target gene in adipose tissue 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 control tissue.
  • expression of a target gene in gonadal white adipose tissue 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 control tissue.
  • expression of a target gene in subcutaneous white adipose tissue 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 liver tissue.
  • expression of a target gene in adipose tissue 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 control tissue.
  • expression of a target gene in gonadal white adipose tissue 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 liver tissue.
  • expression of a target gene in subcutaneous white adipose tissue 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%, FH12501620.1 Attorney Docket: DCY-13025 about 90%, about 95%, about 99% or greater than 99% when compared to target gene expression in liver tissue.
  • contacting or delivering an RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene in adipose tissue (e.g., gWAT and/or scWAT).
  • the reduction in expression of a target gene in adipose tissue is relative to an amount or level of target gene expression in liver tissue contacted with the RNAi oligonucleotide.
  • reduction in expression of a target gene in the adipose tissue (e.g., gWAT and/or scWAT) of a subject 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 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to reduction in expression of the target gene in liver tissue.
  • reduction in expression of a target gene in liver tissue 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 the reduction in expression of the target gene in adipose tissue (e.g., gWAT and/or scWAT).
  • adipose tissue e.g., gWAT and/or scWAT.
  • expression of a target gene in the central nervous system 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 other control tissue.
  • expression of a target gene in the central nervous system 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 liver tissue.
  • contacting or delivering an RNAi oligonucleotide described herein to a cell or a population of cells results in a reduction in expression of a target gene in the central nervous system.
  • the reduction in expression of a target gene in the central nervous system is relative to an amount or level of target gene expression in liver tissue contacted with the RNAi oligonucleotide.
  • reduction in expression of a target gene in the central nervous system of a subject 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 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to reduction in expression of the target gene in liver tissue.
  • reduction in expression of a target gene in liver tissue 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 the reduction in expression of the target gene in the central nervous system.
  • Treatment Methods provides methods for treating a disease, disorder, or condition associated with expression of a target gene in extrahepatic tissue. In some embodiments, the disclosure provides methods for treating a disease, disorder, or condition associated with expression of a target gene in cardiac tissue.
  • the disclosure provides methods for treating a disease, disorder, or condition associated with expression of a target gene in adipose tissue. In some embodiments, the disclosure provides methods for treating a disease, disorder, or condition associated with expression of a target gene in adrenal tissue. In some embodiments, the disclosure provides methods for treating a disease, disorder, or condition associated with expression of a target gene in skeletal muscle tissue. In some embodiments, the disclosure provides methods for treating a disease, disorder, or condition associated with expression of a target gene in the central nervous system. Methods described herein are typically involve administering to a subject a therapeutically effective amount of an RNAi oligonucleotide herein, that is, an amount capable of producing a desirable therapeutic result.
  • a therapeutically acceptable amount may be an amount that can therapeutically treat a disease or disorder.
  • the appropriate dosage for any one subject will depend on certain factors, including the subject ⁇ s size, body surface area, age, the FH12501620.1 Attorney Docket: DCY-13025 composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.
  • a subject is administered any one of the compositions herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra- arterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the brain of a subject).
  • enterally e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally
  • parenterally e.g., subcutaneous injection, intravenous injection or infusion, intra- arterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal
  • an RNAi oligonucleotide herein, or a composition thereof is administered once every year, once every 6 months, once every 4 months, quarterly (once every three months), bi-monthly (once every two months), monthly or weekly.
  • an RNAi oligonucleotide herein, or a composition thereof is administered every week or at intervals of two, or three weeks.
  • an RNAi oligonucleotide herein, or a composition thereof is administered daily.
  • a subject is administered one or more loading doses of an RNAi oligonucleotide herein, or a composition thereof, followed by one or more maintenance doses of the RNAi oligonucleotide, or a composition thereof.
  • the subject to be treated is a human or non-human primate or other mammalian subject.
  • Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.
  • the disclosure provides oligonucleotides for use as a medicament, in particular for use in a method for the treatment of diseases, disorders, and conditions associated with extrahepatic tissue.
  • the disclosure also provides RNAi oligonucleotides for use, or adaptable for use, to treat a subject (e.g., a human) having a disease, disorder or condition associated with expression of a target gene that would benefit from reducing expression of the target gene.
  • the disclosure provides RNAi oligonucleotides for use, or adapted for use, to treat a subject having a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue.
  • RNAi oligonucleotides for use, or adaptable for use, in the manufacture of a medicament or pharmaceutical composition for treating a disease, disorder or condition associated with expression of a target gene in FH12501620.1 Attorney Docket: DCY-13025 extrahepatic tissue.
  • a subject having a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue or is predisposed to the same is selected for treatment with an RNAi oligonucleotide herein.
  • the method comprises selecting an individual having a marker (e.g., a biomarker) for a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue, or predisposed to the same, such as, but not limited to, target mRNA, protein, or a combination thereof.
  • a marker e.g., a biomarker
  • some embodiments of the methods provided by the disclosure include steps such as measuring or obtaining a baseline value for a marker of expression of a target gene in extrahepatic tissue, and then comparing such obtained value to one or more other baseline values or values obtained after the subject is administered the RNAi oligonucleotide to assess the effectiveness of treatment.
  • the disclosure also provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue with an RNAi oligonucleotide provided herein.
  • the disclosure provides methods of treating or attenuating the onset or progression of a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue using the RNAi oligonucleotides provided herein. In some embodiments, the disclosure provides methods to achieve one or more therapeutic benefits in a subject having a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue using the RNAi oligonucleotides provided herein. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of any one or more of the RNAi oligonucleotides provided herein. In some embodiments, treatment comprises reducing expression of a target gene in extrahepatic tissue.
  • the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically. In some embodiments of the methods herein, an RNAi oligonucleotide provided herein, or a pharmaceutical composition comprising the RNAi oligonucleotide, is administered to a subject having a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue such that target gene expression is reduced in the subject, thereby treating FH12501620.1 Attorney Docket: DCY-13025 the subject. In some embodiments, an amount or level of target mRNA is reduced in the subject. In some embodiments, an amount or level of protein encoded by the target mRNA is reduced in the subject.
  • an RNAi oligonucleotide provided herein, or a pharmaceutical composition comprising the RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue such that target gene expression is reduced in the subject 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 prior to administration of the RNAi oligonucleotide or pharmaceutical composition.
  • expression of a target gene in extrahepatic tissue is reduced in the subject 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 subject (e.g., a reference or control subject) not receiving RNAi oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition or treatment.
  • a subject e.g., a reference or control subject
  • an RNAi oligonucleotide herein, or a pharmaceutical composition comprising the RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue such that an amount or level of target mRNA is reduced in the subject 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 the amount or level of target mRNA prior to administration of the RNAi oligonucleotide or pharmaceutical composition.
  • an amount or level of target mRNA is reduced in the subject 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 an amount or level of target mRNA in a subject (e.g., a reference or control subject) not receiving the RNAi oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition or treatment.
  • a subject e.g., a reference or control subject
  • an RNAi oligonucleotide herein, or a pharmaceutical composition comprising the RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of a target gene in FH12501620.1 Attorney Docket: DCY-13025 extrahepatic tissue such that an amount or level of protein encoded by the target gene is reduced in the subject 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 the amount or level of protein encoded by the target gene prior to administration of the RNAi oligonucleotide or pharmaceutical composition.
  • an amount or level of protein encoded by a target gene in extrahepatic tissue is reduced in the subject 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 an amount or level of protein encoded by the target gene in a subject (e.g., a reference or control subject) not receiving the RNAi oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition or treatment.
  • a subject e.g., a reference or control subject
  • an RNAi oligonucleotide herein, or a pharmaceutical composition comprising the RNAi oligonucleotide is administered to a subject having a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue such that an amount or level of target gene activity is reduced in the subject 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 the amount or level of target gene activity prior to administration of the RNAi oligonucleotide or pharmaceutical composition.
  • an amount or level of target gene activity is reduced in the subject 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 an amount or level of target gene activity in a subject (e.g., a reference or control subject) not receiving the RNAi oligonucleotide or pharmaceutical composition or receiving a control oligonucleotide, pharmaceutical composition or treatment.
  • a subject e.g., a reference or control subject
  • an RNAi oligonucleotide comprising a C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a C16-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi FH12501620.1 Attorney Docket: DCY-13025 oligonucleotide comprising a C16-COOH conjugated to a nucleotide at position 28 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a C16-COOH conjugated to a nucleotide in the tetraloop of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in skeletal muscle tissue.
  • an RNAi oligonucleotide comprising a C16-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in skeletal muscle tissue.
  • an RNAi oligonucleotide comprising a PEG4-triz-C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG4-triz-C16-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a C2-triazole-C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a C2-triazole-C16-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a C22-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least FH12501620.1 Attorney Docket: DCY-13025 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a C22-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG4-C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG4-C16-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG 12 -C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG 12 -C16-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a Hexylamine-PEG4-C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a Hexylamine-PEG 4 -C16-COOH conjugated to a nucleotide at position 1 of the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG 2 -C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG2-C16-COOH conjugated to a FH12501620.1 Attorney Docket: DCY-13025 nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in skeletal muscle tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG 2 -C16-COOH conjugated to a nucleotide on the sense strand reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in heart tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG2-C16-COOH conjugated to a nucleotide on the sense strand at position 1 and position 20 reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in adipose tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG2-C16-COOH conjugated to a nucleotide on the sense strand at position 1 and position 20 reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in skeletal muscle tissue relative to target gene knockdown in liver tissue.
  • an RNAi oligonucleotide comprising a PEG 2 -C16-COOH conjugated to a nucleotide on the sense strand at position 1 and position 20 reduces target mRNA expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% more in heart tissue relative to target gene knockdown in liver tissue.
  • Suitable methods for determining target gene expression, an amount or level of target mRNA, an amount or level of protein encoded by the target gene, and/or an amount or level of target gene activity, in the subject, or in a sample from the subject, are known in the art. Further, the Examples set forth herein illustrate exemplary methods for determining target gene expression.
  • target gene expression, an amount or level of target gene mRNA, an amount or level of protein encoded by a target gene, an amount or level of target gene activity, or any combination thereof is reduced in a cell, a population or a group of cells (e.g., an organoid), an organ, blood or a fraction thereof (e.g., plasma), a tissue, a sample (e.g., a biopsy sample), or any other biological material obtained or isolated from the subject.
  • a cell e.g., an organoid
  • an organ e.g., blood or a fraction thereof (e.g., plasma)
  • tissue e.g., a sample
  • sample e.g., a biopsy sample
  • expression of a target gene in extrahepatic tissue is reduced in more than one type of cell, more than one groups of cells, more than one organ, more than one fraction of blood (e.g., plasma FH12501620.1 Attorney Docket: DCY-13025 and one or more other blood fraction(s)), more than one type of tissue, more than one type of sample obtained or isolated from the subject.
  • expression of a target gene in extrahepatic tissue is reduced in one or more of skeletal muscle, cardiac tissue, adipose tissue, and adrenal tissue.
  • expression of a target gene in extrahepatic tissue is reduced in the skeletal muscle. In some embodiments, expression of a target gene in extrahepatic tissue is reduced in the cardiac tissue. In some embodiments, expression of a target gene in extrahepatic tissue is reduced in adipose tissue. In some embodiments, expression of a target gene in extrahepatic tissue is reduced in adrenal tissue.
  • Examples of a disease, disorder or condition associated with expression of a target gene in extrahepatic tissue include, but are not limited to, myopathy, amyotrophic lateral sclerosis, spinal muscular atrophy, multiple sclerosis, muscular dystrophy, lipedema, lipodystrophy, lymphedema, lipomatosis, familial multiple lipomatosis, angiolipomatosis, Dercum disease, multiple symmetric lipomatosis, Proteus syndrome, Cowden Syndrom, Modeling disease, lymphatic leakage, de novo adipogenesis, obesity, and satiety.
  • the target gene in extrahepatic tissue may be a target gene from any mammal, such as a human.
  • kits comprising an RNAi oligonucleotide herein, or a composition thereof, described herein, and instructions for use.
  • the kit comprises an RNAi oligonucleotide herein, or a composition thereof, described herein, and a package insert containing instructions for use of the kit and/or any component thereof.
  • the kit comprises, in a suitable container, a RNAi oligonucleotide herein, or a composition thereof, described herein, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art.
  • the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which the RNAi oligonucleotide herein, or a composition thereof, is placed, and in some instances, suitably aliquoted.
  • the kit contains additional containers into which this component is placed.
  • the kits can also include a means for containing a RNAi oligonucleotide herein, or a composition thereof, and any other reagent in close confinement for commercial sale.
  • Such containers may include injection or blow-molded plastic containers FH12501620.1 Attorney Docket: DCY-13025 into which the desired vials are retained.
  • Containers and/or kits can include labeling with instructions for use and/or warnings.
  • a kit comprises an RNAi oligonucleotide herein, or a composition thereof, described herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the RNAi oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with expression of a target gene expressed extrahepatic tissue in a subject in need thereof.
  • a kit comprises an RNAi oligonucleotide herein, or a composition thereof, described herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the RNAi oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with expression of a target gene expressed in adipose tissue in a subject in need thereof.
  • a kit comprises an RNAi oligonucleotide herein, or a composition thereof, described herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the RNAi oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with expression of a target gene expressed in the adrenal gland in a subject in need thereof.
  • a kit comprises an RNAi oligonucleotide herein, or a composition thereof, described herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the RNAi oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with expression of a target gene expressed in the cardiac tissue in a subject in need thereof.
  • a kit comprises an RNAi oligonucleotide herein, or a composition thereof, described herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the RNAi oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with expression of a target gene expressed in the skeletal muscle in a subject in need thereof.
  • a pharmaceutically acceptable carrier or a pharmaceutical composition comprising the RNAi oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with expression of a target gene expressed in the skeletal muscle in a subject in need thereof.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Q.beta.-replicase amplification RNA polymerase mediated techniques
  • NASBA RNA polymerase mediated techniques
  • homologous nucleic acids of the disclosure are found in Berger, Sambrook, and Ausubel, as well as in Mullis et al., (1987) U.S. Pat. No. 4,683,202; Innis et al., eds. (1990); PCR P ROTOCOLS : A G UIDE TO M ETHODS AND A PPLICATIONS (Academic Press Inc.
  • the term “amount” refers to an absolute amount (e.g., an absolute amount of mRNA or protein), a relative amount (e.g., a relative amount of target mRNA as measured by PCR assay or protein), or a concentration (e.g. a concentration of ligand-conjugated oligonucleotide in a composition), whether the amount referred to in a given instance refers to an absolute amount, concentration, or both, will be clear to the skilled artisan based on the context provided herein.
  • “bicyclic nucleotide” refers to a nucleotide comprising a bicyclic sugar moiety.
  • bicyclic sugar moiety refers to a modified sugar moiety comprising 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.
  • the 4 to 7 membered ring is a sugar.
  • the 4-to-7-member ring FH12501620.1 Attorney Docket: DCY-13025 is a furanosyl.
  • the bridge connects the 2 ⁇ -carbon and the 4 ⁇ -carbon of the furanosyl.
  • complementary refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the two nucleotides to form base pairs with one another.
  • a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another.
  • complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes.
  • two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.
  • deoxyribonucleotide refers to a nucleotide having a hydrogen in place of a hydroxyl at the 2 ⁇ position of its pentose sugar when compared with a ribonucleotide.
  • a modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2 ⁇ position, including modifications or substitutions in or of the sugar, phosphate group or base.
  • double-stranded RNA or “dsRNA” refers to an RNA oligonucleotide that is substantially in a duplex form.
  • the complementary base-pairing of duplex region(s) of a dsRNA oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands.
  • complementary base-pairing of duplex region(s) of a dsRNA formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked.
  • complementary base-pairing of duplex region(s) of a dsRNA is formed from single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together.
  • a dsRNA comprises two covalently separate nucleic acid strands that are fully duplexed with one another.
  • a dsRNA comprises two covalently separate nucleic acid strands that are partially duplexed (e.g., having overhangs at one or both ends).
  • a dsRNA comprises antiparallel sequence of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.
  • duplex in reference to nucleic acids (e.g., oligonucleotides), refers to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides. FH12501620.1 Attorney Docket: DCY-13025
  • excipient refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.
  • loop refers to an unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cells), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).
  • melting temperature or “Tm” means the temperature at which the two strands of a duplex nucleic acid separate. Tm is often used as a measure of duplex stability or the binding affinity of two strands of complementary nucleic acids or portions thereof.
  • modified internucleotide linkage refers to an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, a modified nucleotide is a non-naturally occurring linkage.
  • a modified internucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present.
  • a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.
  • modified nucleotide refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide and thymidine deoxyribonucleotide.
  • a modified nucleotide is a non-naturally occurring nucleotide.
  • a modified nucleotide has one or more chemical modification in its sugar, nucleobase and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.
  • RNAi oligonucleotide refers to a structure of a RNAi oligonucleotide that is characterized by separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity with the antisense strand, and in which at least one of the strands, generally the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed within the at least one strand.
  • oligonucleotide refers to a short nucleic acid (e.g., less than about 100 nucleotides in length).
  • An oligonucleotide may be single stranded (ss) or ds.
  • An oligonucleotide may or may not have duplex regions.
  • an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA or ss siRNA.
  • a double-stranded (dsRNA) is an RNAi oligonucleotide.
  • overhang refers to terminal non-base pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex.
  • an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5 ⁇ terminus or 3 ⁇ terminus of a dsRNA.
  • the overhang is a 3 ⁇ or 5 ⁇ overhang on the antisense strand or sense strand of a dsRNA.
  • phosphate analog refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group.
  • a phosphate analog is positioned at the 5 ⁇ terminal nucleotide of an oligonucleotide in place of a 5 ⁇ - phosphate, which is often susceptible to enzymatic removal.
  • a 5 ⁇ phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include, but are not limited to, 5 ⁇ phosphonates, such as 5 ⁇ methylene phosphonate (5 ⁇ -MP) and 5 ⁇ -(E)-vinylphosphonate (5 ⁇ -VP).
  • an oligonucleotide has a phosphate analog at a 4 ⁇ -carbon position of the sugar (referred to as a “4 ⁇ -phosphate analog”) at a 5 ⁇ - terminal nucleotide.
  • a 4 ⁇ -phosphate analog is 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. See, e.g., US Provisional Patent Application Nos. 62/383,207 (filed on 2 September 2016) and 62/393,401 (filed on 12 September 2016).
  • Other modifications have been developed for the 5 ⁇ end of oligonucleotides (see, e.g., Intl.
  • RNA transcript e.g., target mRNA
  • protein encoded by the target gene e.g., protein encoded by the target gene
  • an appropriate reference e.g., a reference cell, population of cells, sample, or subject
  • an oligonucleotide or conjugate herein e.g., an RNAi oligonucleotide comprising an antisense strand having a nucleotide sequence that is complementary to a nucleotide sequence comprising a target mRNA
  • an oligonucleotide or conjugate herein may result in a decrease in the amount or level of target mRNA, protein encoded by a target gene, and/or target gene activity (e.g., via inactivation and/or degradation of target mRNA by the RNAi pathway) when compared to a cell that is not treated with the double- stranded oligonucleotide.
  • reducing expression refers to an act that results in reduced expression of a target gene.
  • region of complementarity refers to a sequence of nucleotides of a nucleic acid (e.g., a dsRNA) that is sufficiently complementary to an antiparallel sequence of nucleotides to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell, etc.).
  • an oligonucleotide herein comprises a targeting sequence having a region of complementary to a mRNA target sequence.
  • ribonucleotide refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2 ⁇ position.
  • a modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2 ⁇ position, including modifications or substitutions in or of the ribose, phosphate group or base.
  • RNAi oligonucleotide refers to either (a) a dsRNA having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a ss oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.
  • Ago2 Argonaute 2
  • strand refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). In some embodiments, a strand has two free ends (e.g., a 5 ⁇ end and a 3 ⁇ end).
  • subject means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or NHP.
  • “individual” or “patient” may be used interchangeably with “subject.”
  • FH12501620.1 Attorney Docket: DCY-13025
  • “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.
  • targeting ligand refers to a molecule or “moiety” (e.g., a carboxylate, carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and/or that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest.
  • a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue (e.g., extrahepatic tissue) or cell of interest.
  • a targeting ligand selectively binds to a cell surface receptor, such as a fatty acid binding proteins. Accordingly, in some embodiments, a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand and receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell.
  • tetraloop refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides.
  • the increase in stability is detectable as an increase in melting temperature (Tm) of an adjacent stem duplex that is higher than the Tm of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides.
  • Tm melting temperature
  • a tetraloop can confer a Tm of at least about 50°C, at least about 55°C, at least about 56°C, at least about 58°C, at least about 60°C, at least about 65°C or at least about 75°C in 10 mM NaHPO 4 to a hairpin comprising a duplex of at least 2 base pairs (bp) in length.
  • a tetraloop may stabilize a bp in an adjacent stem duplex by stacking interactions.
  • a tetraloop comprises or consists of 3 to 6 nucleotides and is typically 4 to 5 nucleotides.
  • a tetraloop comprises or consists of 3, 4, 5 or 6 nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety).
  • tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., (1990) PROC. NATL. ACAD. SCI. USA 87:8467-71; Antao et al., (1991) NUCLEIC ACIDS RES. 19:5901-05).
  • UUCG UUCG
  • GNRA GNRA
  • GAAA GNRA family of tetraloops
  • CUUG tetraloop Wiese et al., (1990) PROC. NATL. ACAD. SCI. USA 87:8467-71
  • Antao et al. (1991) NUCLEIC ACIDS RES. 19:5901-05).
  • DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)).
  • d(GNNA) family of tetraloops e.g., d(GTTA), the d(GNRA) family of tetraloops
  • the d(GNAB) family of tetraloops e.g., d(CNNG) family of tetraloops
  • d(TNCG) family of tetraloops e.g., d(TTCG)
  • treat or “treating” refers to the act of providing care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., an oligonucleotide herein) to the subject, for purposes of improving the health and/or well-being of the subject with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition.
  • a therapeutic agent e.g., an oligonucleotide herein
  • treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.
  • alkyl or “alkyl group” refers to a fully saturated, straight or branched hydrocarbon chain group, which is attached to the rest of the molecule by a single bond. Alkyls comprising any number of carbon atoms, including but not limited to from 1 to 12 are included. An alkyl comprising up to 12 carbon atoms is a C1-C12 alkyl, an alkyl comprising up to 10 carbon atoms is a C1-C10 alkyl, an alkyl comprising up to 6 carbon atoms is a C1-C6 alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5 alkyl.
  • a C1-C5 alkyl includes C5 alkyls, FH12501620.1 Attorney Docket: DCY-13025 C 4 alkyls, C 3 alkyls, C 2 alkyls and C 1 alkyl (i.e., methyl).
  • a C 1 -C 6 alkyl includes all moieties described above for C 1 -C 5 alkyls but also includes C 6 alkyls.
  • a C 1 -C 10 alkyl includes all moieties described above for C 1 -C 5 alkyls and C 1 -C 6 alkyls, but also includes C 7 , C 8 , C 9 and C 10 alkyls.
  • a C 1 -C 12 alkyl includes all the foregoing moieties, but also includes C 11 and C12 alkyls.
  • Non-limiting examples of C1-C12 alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n– nonyl, n-decyl, n-undecyl, and n-dodecyl.
  • Non-limiting examples of C 2 -C 12 alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1- pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5- hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2- octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3- nonenyl, 4-nonen
  • alkyl group can be optionally substituted.
  • alkenylene or “alkenylene chain” refers to an unsaturated, straight or branched divalent hydrocarbon chain radical having one or more olefins and from two to twelve carbon atoms.
  • C2-C12 alkenylene include ethenylene, propenylene, n-butenylene, and the like.
  • the alkenylene chain is attached to the rest of the molecule through a single bond and to a radical group (e.g., those described herein) through a single bond.
  • the points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain.
  • alkenylene chain can be optionally substituted.
  • Alkynyl or alkynyl group refers to a straight or branched hydrocarbon chain having from two to twelve carbon atoms and having one or more carbon-carbon triple bonds. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl group comprising any number of carbon atoms from 2 to 12 are included.
  • An alkynyl group comprising up to 12 carbon atoms is a C2-C12 alkynyl
  • an alkynyl comprising up to 10 carbon atoms is a C 2 -C 10 alkynyl
  • an alkynyl group comprising up to 6 carbon atoms is a C 2 -C 6 alkynyl
  • an alkynyl comprising up to 5 carbon atoms is a C2-C5 alkynyl.
  • a C2-C5 alkynyl includes C5 alkynyls, C4 alkynyls, C3 alkynyls, and C2 alkynyls.
  • a C2-C6 alkynyl includes all moieties described above for C2-C5 alkynyls but also includes C6 alkynyls.
  • a C2-C10 alkynyl includes all moieties described above for C2-C5 alkynyls and C2-C6 alkynyls, but also includes C7, C8, C9 and C10 alkynyls.
  • a C2-C12 alkynyl includes all the foregoing moieties, but also includes C11 and C12 alkynyls.
  • Non-limiting examples of C2-C12 alkenyl include ethynyl, propynyl, butynyl, pentynyl and the like.
  • alkyl group can be optionally substituted.
  • Alkynylene or “alkynylene chain” refers to an unsaturated, straight or branched divalent hydrocarbon chain radical having one or more alkynes and from two to twelve carbon atoms.
  • Non-limiting examples of C 2 -C 12 alkynylene include ethynylene, propynylene, n-butynylene, and the like.
  • the alkynylene chain is attached to the rest of the molecule through a single bond and to a radical group (e.g., those described herein) through a single bond.
  • alkynylene chain The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group can be through any two carbons within the chain having a suitable valency. Unless stated otherwise, an alkynylene chain can be optionally substituted.
  • FH12501620.1 Attorney Docket: DCY-13025 “Alkoxy” refers to a group of the formula -OR a where R a is an alkyl, alkenyl or alknyl as defined above containing one to twelve carbon atoms. Unless stated otherwise, an alkoxy group can be optionally substituted.
  • Cycloalkyl refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon group consisting solely of carbon and hydrogen atoms, which can include fused or bridged ring systems, having from three to twenty carbon atoms, preferably having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond.
  • Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Polycyclic cycloalkyl groups include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkyl group can be optionally substituted.
  • Aryl refers to a hydrocarbon ring system group comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring.
  • the aryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems.
  • Aryl groups include, but are not limited to, aryl groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene.
  • aryl is meant to include aryl groups that are optionally substituted.
  • Heterocyclyl refers to a stable 3- to 20-membered ring group which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.
  • the heterocyclyl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems.
  • the nitrogen, carbon or sulfur atoms in the heterocyclyl group can be optionally oxidized, the nitrogen atom can be optionally quaternized.
  • the heterocyclyl group can be partially or fully saturated.
  • heterocyclyl groups include, but are not limited to, dioxolanyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.
  • heteroaryl refers to a 5- to 20-membered ring system group comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring.
  • the heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group can be optionally oxidized; the nitrogen atom can be optionally quaternized.
  • Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furany
  • a heteroaryl group can be optionally substituted.
  • carboxyl refers to radical group -COOH, or a charged form thereof, including carboxylate (e.g., -COO-). Therefore, any reference herein to a carboxyl group, such as a carboxylated ligand, also include the changed forms of said group.
  • substituted means any of the above groups wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N- oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups
  • “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups FH12501620.1 Attorney Docket: DCY-13025 such as imines, oximes, hydrazones, and nitriles.
  • Rg and Rh are the same or different and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl.
  • “Substituted” further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl group.
  • a double-stranded oligonucleotide comprising: (i) an antisense strand of 15 to 30 nucleotides in length, a sense strand of 13 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in an extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and (ii) one or more ligands, each ligand comprising one or more -L-Y-(CO2H)n groups, or a charged form thereof, wherein L is a linker, Y is alkylene, alkenylene, or alkynylene, and n is 1-6, wherein when Y is alkylene, L comprises -O(CH 2
  • a double-stranded oligonucleotide comprising: (i) a double-stranded oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length, a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand has a region of complementarity to a target sequence in a target mRNA in extrahepatic tissue, and wherein the region of complementarity is at least 15 contiguous nucleotides in length; and FH12501620.1 Attorney Docket: DCY-13025 (ii) one or more ligands, each ligand comprising one or more C 6-24 alkylene-(CO 2 H) n , or a charged form thereof, conjugated to a nucleotide of the sense strand, wherein n is 1-6, wherein knock down of the target mRNA in the extrahepatic tissue is greater than knock down of the target mRNA in liver tissue.
  • Embodiment I-3 The double-stranded oligonucleotide of Embodiment I-2, wherein each ligand comprises a linker (L), wherein L is conjugated to the one or more C6-24 alkylene- CO2H.
  • L comprises a bivalent or trivalent C1-C50 alkylene, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1-C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2-, -S(O)2N(C1-C4 alkyl)-, -S(O)2N(cycloalkyl)-, - N(H)C(O)-, -N(C1-C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1-C4 alkyl), - C
  • Embodiment I-5 The double-stranded oligonucleotide of Embodiment I-4, wherein 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(H)C(O)-, -N(C 1 - C 4 alkyl)C(O)-, -O-, or heteroaryl.
  • Embodiment I-6 The double-stranded oligonucleotide of Embodiment I-4 or 5, wherein the heteroaryl is a triazolyl.
  • Embodiment I-7 The double-stranded oligonucleotide of Embodiment I-6, wherein the triazolyl Embodiment I-8.
  • Embodiment I-9. The double-stranded oligonucleotide of any one of Embodiments I-1-8, wherein Y is an C6-24 alkylene or C6-24 alkenylene.
  • Embodiment I-10 The double-stranded oligonucleotide of any one of Embodiments I-1-9, wherein Y is a C6-24 alkenylene.
  • Embodiment I-12. The double-stranded oligonucleotide of any one of Embodiments I-1- 10, wherein the alkenylene comprises from 1-6 olefinic bonds.
  • Embodiment I-13. The double-stranded oligonucleotide of any one of Embodiments I-1- 10, wherein the alkynylene comprises from 1-6 acetylenic bonds.
  • FH12501620.1 Attorney Docket: DCY-13025 Embodiment I-14.
  • Embodiment I-41. The double-stranded oligonucleotide of any one of Embodiments I-35- 39, wherein the sense strand is 20 nucleotides in length, and wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2, between positions 18 and 19, and between positions 19 and 20, wherein positions are numbered 1-20 from 5 ⁇ to 3 ⁇ .
  • Antisense A was prepared by solid-phase synthesis (LCMS found mass ⁇ ).
  • Synthesis of Duplex B Conjugated Sense B was synthesized through a two-step post solid phase conjugation approach.
  • Step 1- Conjugation of Azido-PEG4 Handle: In Eppendorf tube 1, a solution of Azido-PEG4-NHS ester (4.25 mg, 0.011 mmol) in 562 ⁇ L DMF was prepared at rt. In Eppendorf tube 2, oligo Sense B1 (22.5 mg, 0.0018 mmol) was dissolved in 562 ⁇ L water and treated with DIPEA (1.95 ⁇ L, 0.011 mmol).
  • Duplex B was prepared using the same procedures as described for the annealing of Duplex A.
  • Synthesis of Duplex C FH12501620.1 Attorney Docket: DCY-13025 Conjugated Sense C was synthesized through a two-step post solid phase conjugation approach similar to Conjugated Sense B.
  • Step 1- Attachment of Azido-PEG4 Handle In Eppendorf tube 1, a solution of Azido-PEG4-NHS ester (8.5 mg, 0.022 mmol) in 1125 ⁇ L DMF was prepared at rt.
  • Conjugated Sense E was synthesized through a two-step post solid phase conjugation approach similar to Conjugated Sense C.
  • Step 1- Attachment of Azido-PEG4 Handle In Eppendorf tube 1, a solution of Azido- PEG4-NHS ester (8.5 mg, 0.022 mmol) in 1125 ⁇ L DMF was prepared at rt.
  • oligo Sense C1 In Eppendorf tube 2, oligo Sense C1 (45 mg, 0.0036 mmol) was dissolved in 1125 ⁇ L water and treated with DIPEA (3.9 ⁇ L, 0.022 mmol).
  • Step 2- Conjugation to Azido-PEG4 Handle: FH12501620.1 Attorney Docket: DCY-13025 In Eppendorf tube 1, a 1:1 mixture of water and DMA (360 ⁇ L) was degassed under nitrogen gas for 10 minutes. Next 20 mg (1 eq, 0.0016 mmol) of Conjugated Sense C2 and 1.7 ⁇ L DIPEA (6eq, 0.0096 mmol) were added to the mixture of water and DMA in Eppendorf tube 1, followed by addition of 2.682 mg Propargyl-PEG3-acid (6 eq, 0.0096 mmol) to Eppendorf tube 1. The resulting mixture was kept under Nitrogen gas for another 10 minutes to remove any dissolved oxygen.
  • Duplex E was prepared using the same procedures as described for the annealing of Duplex A. Synthesis of Duplex F and Duplex G: FH12501620.1 Attorney Docket: DCY-13025 Conjugated Sense F and G were synthesized through post solid-phase conjugation approach substantially similar to Conjugated Sense E. Post-synthetic conjugation was realized through two step Amide reaction followed by Cu-catalyzed alkyne-azide cycloaddition reaction. 6.32 mg of Conjugated Sense F was obtained in 31.6% yield (LCMS found mass 12627). 12.56 mg of Conjugated Sense G was obtained in 62.8% yield (LCMS found mass 12779).
  • Duplex F and Duplex G were prepared using the same procedures as described for the annealing of Duplex A.
  • Synthesis of Duplex H Conjugated Sense H was synthesized through a one-step post-syntenic conjugation approach.
  • a solution of Palmitic acid 1.5 mg, 0.0053 mmol
  • DMA 495 ⁇ L
  • HATU 2.01 mg, 0.0053 mmol
  • Eppendorf tube 2 a solution of oligo Sense C1 (13 mg, 0.00106 mmol) in H2O (55 ⁇ L) was treated with DIPEA (1.11 ⁇ L, 0.00636 mmol).
  • oligonucleotides were synthesized using 2’-modified nucleoside phosphoramidites, such as 2'-F or 2'-OMe, and 2'-diethoxymethanol linked Docosanoic acid (C22) fatty acid amide nucleoside phosphoramidites. Oligonucleotide synthesis was conducted on a solid support in the 3' to 5' direction using a standard oligonucleotide synthesis protocol. In these efforts, 5-ethylthio-1H-tetrazole (ETT) was used as an activator for the coupling reaction.
  • ETT 5-ethylthio-1H-tetrazole
  • Eppendorf tube 1 a solution of hexadecanedioic acid (2.51 mg, 0.00879 mmol) in DMA FH12501620.1 Attorney Docket: DCY-13025 (800 ⁇ L) was treated with HATU (5.6 mg, 0.01465 mmol) at rt.
  • Eppendorf tube 2 a solution of oligo Sense K (20 mg, 0.00293 mmol) in 200 ⁇ L H 2 O was treated with DIPEA (3 ⁇ L, 0.01758 mmol). The solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed using Thermomixer at rt.
  • reaction mixture was diluted with 5 mL of water and purified by revers phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O.
  • the product fractions were concentrated under reduced pressure using Genevac.
  • the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra-15 Centrifugal (3K).
  • Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense K (15.14 mg, 75.7% yield) (LCMS found mass 7084).
  • Step 1- Attachment of Amino-PEG4 Handle In Eppendorf tube 1, a solution of Fmoc-N-amido-PEG4-NHS Ester (5.144 mg, 0.00880 mmol) was dissolved in DMA (500 ⁇ L) and treated with DIPEA (1.531 ⁇ L, 0.00879 mmol). In Eppendorf tube 2, a solution of oligo Sense K1 (20 mg, 0.00293 mmol) in 500 ⁇ L H2O was treated with DIPEA (1.531 ⁇ L, 0.00879 mmol). The solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed for 1 hour using Thermomixer at 40°C.
  • the combined residual solvent was dialyzed against water (2 X), saline (2 X), and water (2 X) using Pierce Thermo Fisher 3k filters.
  • the filter membrane was washed with water (3 X 2 mL) and with a last wash of 20% FH12501620.1 Attorney Docket: DCY-13025 ethanol in water (20 ⁇ L).
  • the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense M1 (15.14 mg, 75.7% yield).
  • Step 2- Conjugation to Amino-PEG4 Handle
  • a solution of hexadecanedioic acid (6.137 mg, 0.02143 mmol) was dissolved in DMA (450 ⁇ L) and treated with HATU (8.148 mg, 0.002143 mmol) and DIPEA (1.9 ⁇ L, 0.00107 mmol) at rt.
  • HATU 8.148 mg, 0.002143 mmol
  • DIPEA 1.9 ⁇ L, 0.00107 mmol
  • the solution in Eppendorf tube 1 was then added to the Eppendorf tube 2 and mixed for 30 minutes using Thermomixer at 40°C. After the reaction was completed indicated by LC-MS analysis, the reaction mixture was diluted with 5 mL of water and purified by revers phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O. The product fractions were concentrated under reduced pressure using Genevac. The combined residual solvent was dialyzed against water (2 X), saline (2 X), and water (2 X) using Pierce Thermo Fisher 3k filters. The Amicon membrane was washed with water (3 X 2 mL) and with a last wash of 20% ethanol in water (20 ⁇ L).
  • Conjugated Sense M was synthesized through a two-step post-syntenic conjugation approach substantially similar to Conjugated Sense M. 2 mg of Conjugated Sense N was obtained in 10% yield (LCMS found mass 7683). Sense N was annealed to Antisense B to yield Duplex N using the same procedures as described for the annealing of Duplex A.
  • Step 1 Attachment of Azide Handle: In Eppendorf tube 1, a solution of 3-azidopropanoic acid (1.4 mg, 0.015 mmol) in DMA (500 ⁇ L) was treated with HATU ( 5.6 mg, 0.015 mmol) at rt. In Eppendorf tube 2, oligo Sense K1 (20 mg, 0.003 mmol) was dissolved in 500 ⁇ L water and treated with DIPEA (3 ⁇ L, 0.018 mmol).
  • the combined solvents were purified by revers phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O.
  • the product fractions were concentrated under reduced pressure using Genevac.
  • the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra-15 Centrifugal (3K).
  • the Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense O (1.01 mg, yield: 4.8%) (LCMS found mass 7193).
  • Sense O was annealed to Antisense B to yield Duplex O using the same procedures as described for the annealing of Duplex A.
  • FH12501620.1 Attorney Docket: DCY-13025
  • Synthesis of Duplex P Conjugated Sense P was synthesized through a two-step post-syntenic conjugation approach.
  • oligo Sense K1 (20 mg, 0.002934 mmol) was dissolved in 500 ⁇ L water and treated with DIPEA (3 ⁇ L, 0.017604 mmol). Next, the solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed using Thermomixer at 35 ⁇ C. After 1 hour, the reaction was indicated complete by LC-MS analysis. The reaction mixture was diluted with 10 mL of water and then dialyzed against water (2X) using Amicon® Ultra-15 Centrifugal (10K).
  • the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra-15 Centrifugal (3K).
  • the Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense P (2.2 mg, yield: 10%) (LCMS found mass 7369).
  • Sense P was annealed to Antisense B to yield Duplex P using the same procedures as described for the annealing of Duplex A.
  • Step 1 Attachment of Azido-PEG4 Handle: In Eppendorf tube 1, a solution of Azido-PEG4-NHS ester (6.8 mg, 0.017604 mmol) in 500 ⁇ L DMF was prepared at rt. In Eppendorf tube 2, oligo Sense Q1 (20 mg, 0.002934 mmol) was dissolved in 500 ⁇ L water and treated with DIPEA (3 ⁇ L, 0.017604 mmol).
  • Step 2- Conjugation to Azido-PEG4 Handle: FH12501620.1 Attorney Docket: DCY-13025 In Eppendorf tube 1, a 1:1 mixture of water and DMA (360 ⁇ L) was degassed under nitrogen gas for 10 minutes. Next 20 mg (1 eq, 0.00282 mmol) of Conjugated Sense Q2 and 3 ⁇ L DIPEA (6eq, 0.0169 mmol) were added to the mixture of water and DMA in Eppendorf tube 1, followed by addition of 4.7 mg 17-Octadecynoic acid (6 eq, 0.0096 mmol) to Eppendorf tube 1. The resulting mixture was kept under Nitrogen gas for another 10 minutes to remove any dissolved oxygen.
  • Eppendorf tube 2 4.6 mg CuBr.SCH3 (8 eq, 0.02257 mmol) was dissolved in 240 ⁇ L of previously degassed acetonitrile. Next, the solution in Eppendorf tube 2 was quickly added to Eppendorf tube 1 and mixed using Thermomixer at 35 °C for 3 hours. After the reaction was indicated complete by LC-MS analysis, the reaction mixture was diluted with 15 mL EDTA 0.5 M (pH 8) in a 50mL falcon tube and stirred for 15 minutes at room temperature. Mixture was then dialyzed against water (2X) using Amicon® Ultra-15 Centrifugal (3K).
  • the combined solvents were purified by revers phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H 2 O.
  • the product fractions were concentrated under reduced pressure using Genevac.
  • the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra-15 Centrifugal (3K).
  • the Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense Q (1 mg, yield: 5%) (LCMS found mass 7445).
  • Sense Q was annealed to Antisense B to yield Duplex Q using the same procedures as described for the annealing of Duplex A.
  • FH12501620.1 Attorney Docket: DCY-13025
  • Synthesis of Duplex R Conjugated Sense R was synthesized through a two-step post-syntenic conjugation approach.
  • Step 1- Attachment of Azido-PEG4 Handle In Eppendorf tube 1, a solution of Azido-PEG4-NHS ester (6.8 mg, 0.017604 mmol) in 500 ⁇ L DMF was prepared at rt.
  • Sense K1 (20 mg, 0.002934 mmol) was dissolved in 500 ⁇ L water and treated with DIPEA (3 ⁇ L, 0.017604 mmol). Next, the solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed using Thermomixer at 35 ⁇ C. After 1 hour, the reaction was indicated complete by LC-MS analysis. The reaction mixture was diluted with 10 mL of water and then dialyzed against water (2X) using Amicon® Ultra- 15 Centrifugal (10K).
  • the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra-15 Centrifugal (3K).
  • the Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense R (2.2 mg, yield: 10%) (LCMS found mass 7331).
  • Sense R was annealed to Antisense B to yield Duplex R using the same procedures as described for the annealing of Duplex A.
  • Step 1 Attachment of Azido-PEG4 Handle: In Eppendorf tube 1, a solution of Azido-PEG4-NHS ester (12.7 mg, 0.03252 mmol) in 375 ⁇ L DMF was prepared at rt. In Eppendorf tube 2, Sense S1 (15 mg, 0.002168 mmol) was dissolved in 375 ⁇ L water and treated with DIPEA (6 ⁇ L, 0.0325 mmol).
  • Step 2- Conjugation to Azido-PEG4 Handle: In Eppendorf tube 1, a 1:4 mixture of water and DMA (750 ⁇ L) was degassed under nitrogen gas for 10 minutes. Next 12.5 mg (1 eq, 0.001674mmol) of Conjugated Sense S2 and 4.4 ⁇ L FH12501620.1 Attorney Docket: DCY-13025 DIPEA (6eq, 0.02512 mmol) were added to the mixture of water and DMA in Eppendorf tube 1, followed by addition of 7 mg Octadec-1-yne (6 eq, 0.02512 mmol) to Eppendorf tube 1. The resulting mixture was kept under Nitrogen gas for another 10 minutes to remove any dissolved oxygen.
  • the combined solvents were purified by revers phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O.
  • the product fractions were concentrated under reduced pressure using Genevac.
  • the combined residual solvent was dialyzed against water (1 X), saline (1 X), and water (3 X) using Amicon® Ultra-15 Centrifugal (3K).
  • the Amicon membrane was washed with water (3 X 2 mL) and the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense S (4.49 mg, 25.8% yield) (LCMS found mass 7966).
  • Sense S was annealed to Antisense B to yield Duplex S using the same procedures as described for the annealing of Duplex A.
  • Synthesis of Duplex T Conjugated Sense T was synthesized through a two-step post-syntenic conjugation approach substantially similar to Conjugated Sense S. 8.41 mg of Conjugated Sense T was obtained in 48.4% yield (LCMS found mass 8026).
  • Sense T was annealed to Antisense B to yield Duplex T using the same procedures as described for the annealing of Duplex A.
  • Synthesis of Duplex U FH12501620.1 Attorney Docket: DCY-13025 Conjugated Sense U was synthesized utilizing a two-step conjugation approach.
  • Step 1- Attachment of Azido-PEG4 Handle In Eppendorf tube 1, a solution of Azido-PEG4-NHS ester (12.7 mg, 0.03252 mmol) in 375 ⁇ L DMF was prepared at rt. In Eppendorf tube 2, Sense U1 (15 mg, 0.002168 mmol) was dissolved in 375 ⁇ L water and treated with DIPEA (6 ⁇ L, 0.0325 mmol). Next, the solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed using Thermomixer at 35 ⁇ C. After 1 hour, the reaction was indicated complete by LC-MS analysis.
  • oligonucleotides were synthesized using 2’-modified nucleoside phosphoramidites, such as 2'-F or 2'-OMe, and 2'-diethoxymethanol linked hexadecanoic acid (C16) fatty acid amide nucleoside phosphoramidites. Oligonucleotide synthesis was conducted on a solid support in the 3' to 5' direction using a standard oligonucleotide synthesis protocol. In these efforts, 5-ethylthio-1H-tetrazole (ETT) was used as an activator for the coupling reaction.
  • ETT 5-ethylthio-1H-tetrazole
  • Step 1- Attachment of Amino-PEG4 Handle In Eppendorf tube 1, a solution of Fmoc-N-amido-PEG4-NHS Ester (285 mg, 0.488 mmol, 6.0 equiv) was dissolved in DMF (20 mL). In Eppendorf tube 2, a solution of oligo Sense X1 (500 mg, 0.0813 mmol, 1 equiv) in 5 mL H 2 O was treated with DIPEA (142 ⁇ L, 0.8127 mmol, 10 equiv). The solution in Eppendorf tube 1 was added to the Eppendorf tube 2 and mixed for 1 hour using Thermomixer at 35°C.
  • the crude was then dialyzed against water (2 X) using Pierce Thermo Fisher 3k filters to remove excess of Fmoc-N-amido-PEG4-NHS Ester.
  • the crude was then removed from filters and deprotected using water and Piperidine mixture (20 mL water: 3 mL Piperidine) and mixed for 1 h using Thermomixer at 35°C.
  • the reaction was then diluted with 10 mL of water and purified by reverse phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H2O.
  • the product fractions were concentrated under reduced pressure using Genevac.
  • the combined residual solvent was dialyzed against water (2 X), saline (2 X), and water (2 X) using Pierce Thermo Fisher 3k filters.
  • the filter membrane was washed with water (3 X 2 mL) and with a last wash of 20% ethanol in water (2000 ⁇ L).
  • the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense X2 (550 mg, 100% yield).
  • Step 2- Conjugation to Amino-PEG4 Handle FH12501620.1
  • a solution of docosanedioic acid (290 mg, 0.7813 mmol, 10 equiv) was dissolved in DMA (17.5 mL) and treated with HATU (298 mg, 0.7813 mmol, 10 equiv) and DIPEA (140 ⁇ L, 0.7813 mmol, 10 euqiv) at rt.
  • the combined residual solvent was dialyzed against water (2 X), saline (2 X), and water (2 X) using Pierce Thermo Fisher 3k filters.
  • the Amicon membrane was washed with water (3 X 2 mL) and with a last wash of 20% ethanol in water (20 ⁇ L).
  • the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense X (222 mg, 40% yield) (LCMS found mass 6751).
  • Sense X was annealed to Antisense B to yield Duplex X using the same procedures as described for the annealing of Duplex A.
  • Step 2- Conjugation to Amino-PEG4 Handle
  • Eppendorf tube 1 a solution of hexadecanedioic acid (95 mg, 0.3313 mmol, 10 equiv) was dissolved in DMA (17.5 mL) and treated with HATU (126 mg, 0.3313 mmol, 10 equiv) and DIPEA (70 ⁇ L, 0.3313 mmol, 10 euqiv) at rt.
  • the combined residual solvent was dialyzed against water (2 X), saline (2 X), and water (2 X) using Pierce Thermo Fisher 3k filters.
  • the Amicon membrane was washed with water (3 X 2 mL) and with a last wash of 20% ethanol in water (20 ⁇ L).
  • the combined solvents were then lyophilized to afford an amorphous white solid of Conjugated Sense AB (5.0 mg, 0.00068 mmol, 48% yield) (LCMS found mass 7417).
  • Sense AB was annealed to Antisense B to yield Duplex AB using the same procedures as described for the annealing of Duplex A.
  • Step 2 16,16-di-tert-butyl 1-methyl heptadecane-1,16,16-tricarboxylate (ABd) To a 1-dram vial was added NaH (4 mg, 1.1 equiv), The vial was evacuated with vacuum and back filled with nitrogen three times, then cooled in ice/water bath.
  • Step 3 18-(tert-butoxy)-17-(tert-butoxycarbonyl)-17-methyl-18-oxooctadecanoic acid (ABe)
  • THF 1616-di-tert-butyl 1-methyl heptadecane-1,16,16- tricarboxylate
  • MeOH 0.5 mL
  • water 0.5 mL
  • LiOH 32 mg, 10 equiv
  • the mixture was stirred at r.t. for 3 h. After 3 h, the mixture was diluted with DCM, acidified with citric acid until pH ⁇ 2, and washed with sat. ammonium chloride.
  • Step 4&5 2-(16-((2,5-dioxopyrrolidin-1-yl)oxy)-16-oxohexadecyl)-2-methylmalonic acid (ABa)
  • ABa 2-(16-((2,5-dioxopyrrolidin-1-yl)oxy)-16-oxohexadecyl)-2-methylmalonic acid
  • ABa 2-(16-((2,5-dioxopyrrolidin-1-yl)oxy)-16-oxohexadecyl)-2-methylmalonic acid
  • Step 2- Conjugation to Amino-PEG4 Handle
  • a solution of 16-sulfohexadecanoic acid (4.77 mg, 0.0142 mmol, 10 equiv) was dissolved in DMF (500 ⁇ L) and treated with HATU (4.29 mg, 0.0113 mmol, 8 equiv) and DIPEA (2.5 ⁇ L, 0.0142 mmol, 10 equiv) at rt and mixed for 15 minutes.
  • Eppendorf tube 2 a solution of Conjugated Sense M1 (10.0 mg, 0.00142 mmol, 1 equiv) in 100 ⁇ L H2O was treated with DIPEA (2.5 ⁇ L, 0.0142 mmol, 10 equiv). The solution in Eppendorf tube 1 was then added to the Eppendorf tube 2 and mixed for 30 minutes using Thermomixer at 40°C. After the reaction was completed indicated by LC-MS analysis, the reaction mixture was diluted with 5 mL of water and purified by reverse phase XBridge C18 column using a 5-95% gradient of 100 mM TEAA in ACN and H 2 O. The product fractions were concentrated under reduced pressure using GeneVac.

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Abstract

L'invention concerne des conjugués oligonucléotide d'ARNi-ligand qui inhibent ou réduisent l'expression de gènes cibles. L'invention concerne également des compositions les comprenant et leurs utilisations, en particulier leurs utilisations pour le traitement de maladies, de troubles et/ou d'états pathologiques extra-hépatiques.
PCT/US2024/045600 2023-09-08 2024-09-06 Conjugués d'oligonucléotides d'arni WO2025054459A1 (fr)

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