WO2025259747A2 - Compositions d'arni de protéine kinase myotonique de dystrophie (dmpk) et leurs procédés d'utilisation - Google Patents

Compositions d'arni de protéine kinase myotonique de dystrophie (dmpk) et leurs procédés d'utilisation

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Publication number
WO2025259747A2
WO2025259747A2 PCT/US2025/033136 US2025033136W WO2025259747A2 WO 2025259747 A2 WO2025259747 A2 WO 2025259747A2 US 2025033136 W US2025033136 W US 2025033136W WO 2025259747 A2 WO2025259747 A2 WO 2025259747A2
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acceptable salt
pharmaceutically acceptable
dsrna agent
nucleotide
group
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WO2025259747A3 (fr
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Karyn SCHMIDT
Mark K. SCHLEGEL
Adam CASTORENO
James D. MCININCH
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Alnylam Pharmaceuticals Inc
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Alnylam Pharmaceuticals Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--
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    • C12N2310/34—Spatial arrangement of the modifications
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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
    • C12N2310/35—Nature of the modification
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    • 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

  • DMPK dystrophy myotonic protein kinase
  • MT-PK myotonin-protein kinase
  • MT-PK myotonin-protein kinase
  • DM1 Myotonic dystrophy
  • CTG cytosine thymine guanine
  • DM1 is a multisystem disorder characterized by myotonia, muscular dystrophy, cataracts, hypogonadism, frontal balding, cardiac conduction defects, endocrine abnormalities, and iridescent cataracts, 25 although the clinical features of DM1 and the severity of disease vary with the number of repeats: normal individuals have 5 to 37 repeats, mildly affected persons have 50 to 150 repeats, patients with classic DM1 have 100 to 1,000 repeats, and those with onset at birth can have more than 2,000 repeats. Effective therapies and treatments for DM1 are currently not available and, instead, 30 treatments and therapies focus on managing symptoms and improving quality of life.
  • RNA-induced silencing complex RISC
  • the DMPK gene may be within a cell, e.g., a cell within a subject, such as a human subject.
  • the present disclosure also provides methods of using the iRNA compositions of the disclosure for inhibiting the expression of a DMPK gene, and/or for treating a subject who would benefit from inhibiting or reducing the expression of an DMPK gene, e.g., a subject suffering or prone 5 to suffering from an DMPK-associated disorder, e.g., Myotonic dystrophy (DM) type 1 (DM1).
  • DM Myotonic dystrophy
  • the disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a dystrophy myotonic protein kinase (DMPK) gene in a cell, or a pharmaceutically acceptable salt thereof, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 18, 10 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than three nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-3.
  • dsRNA double stranded ribonucleic acid
  • DMPK dystrophy myotonic protein kinase
  • the disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a dystrophy myotonic protein kinase (DMPK) gene in a cell, or a pharmaceutically acceptable salt thereof, comprising a sense strand and an antisense strand forming a 15 double stranded region; wherein the sense strand comprises at least 15, e.g., 15, 16, 18, 18, 19, 20, or 21, contiguous nucleotides differing by no more than three, e.g., 3, 2, 1, or 0, nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15, e.g., 15, 16, 18, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than three, e.g., 3, 2, 1, or 0, nucleotides from the corresponding portion of the nucleotide sequence of SEQ ID NO:2; at least one 20 alpha-v-beta
  • the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a dystrophy myotonic protein kinase (DMPK) gene in a cell, or a 25 pharmaceutically acceptable salt thereof, comprising a sense strand and an antisense strand forming a double stranded region; wherein the antisense strand comprises at least 15, e.g., 15, 16, 18, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than three, e.g., 3, 2, 1, or 0, nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-3; at least one alpha- v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least 30 one strand; and at least one in vivo delivery enhancing moiety conjugated to one or more internal positions on at least one strand.
  • dsRNA double strande
  • the sense strand comprises at least 15, e.g., 15, 16, 18, 18, 19, 20, or 21, contiguous nucleotides differing by no more than three, e.g., 3, 2, 1, or 0, nucleotides from any one of the sense strand nucleotide sequences in any one of Tables 2-3.
  • the sense strand comprises at least 15, e.g., 15, 16, 18, 18, 19, 20, or 21, contiguous nucleotides differing by no more than two nucleotides from any one of the sense strand nucleotide sequences in any one of Tables 2-3 and the antisense strand comprises at least 15, e.g., 15, 2 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 16, 18, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than two nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-3.
  • the sense strand comprises at least 15, e.g., 15, 16, 18, 18, 19, 20, or 21, contiguous nucleotides differing by no more than one nucleotide from any one of the sense strand 5 nucleotide sequences in any one of Tables 2-3 and the antisense strand comprises at least 15, e.g., 15, 16, 18, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than one nucleotide from any one of the antisense strand nucleotide sequences in any one of Tables 2-3.
  • the sense strand comprises a nucleotide sequence selected from the group consisting of any one of the sense strand nucleotide sequences in any one of Tables 2-3 and the 10 antisense strand comprises a nucleotide sequence selected from the group consisting of any one of the antisense strand nucleotide sequences in any one of Tables 2-3.
  • the dsRNA agent, or a pharmaceutically acceptable salt thereof comprises at least one nucleotide comprising a nucleotide modification.
  • all of the nucleotides of the sense strand comprise a nucleotide 15 modification; all of the nucleotides of the antisense strand comprise a nucleotide modification; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a nucleotide modification.
  • At least one of the nucleotide modifications is selected from the group consisting of a deoxy-nucleotide modification, a 3’-terminal deoxythimidine (dT) nucleotide 20 modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy nucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2’-amino nucleotide modification, a 2’-O-allyl nucleotide modification, 2’-C-alkyl nucleotide modification, 2’-hydroxly nucleotide modification, a 2’- 25 methoxyethyl nucleotide modification, a 2’-O-alkyl
  • At least one of the modified nucleotides is selected from the group 35 consisting of LNA, HNA, CeNA, 2’-methoxyethyl, 2’-O-alkyl, 2’-O-allyl, 2’-C- allyl, 2’-fluoro, 2’- deoxy, 2’-hydroxyl, and glycol; and combinations thereof.
  • at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, 3 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO a 2'-deoxy-modified nucleotide, a glycol modified nucleotide (GNA), a nucleotide comprising a 2’ phosphate, a nucleotide comprising a phosphorothioate group, and a vinyl-phosphonate nucleotide; and combinations thereof.
  • GAA glycol modified nucleotide
  • At least one of the modified nucleotides is a nucleotide modified with a 5 thermally destabilizing nucleotide modification, e.g., selected from the group consisting of an abasic modification; a mismatch with the opposing nucleotide in the duplex; a destabilizing sugar modification, a 2’-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).
  • a 5 thermally destabilizing nucleotide modification e.g., selected from the group consisting of an abasic modification; a mismatch with the opposing nucleotide in the duplex; a destabilizing sugar modification, a 2’-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).
  • a 5 thermally destabilizing nucleotide modification
  • the dsRNA agent, or a pharmaceutically acceptable salt thereof, further 10 comprises a phosphate or phosphate mimic at the 5’-end of the antisense strand, e.g., a 5’-vinyl phosphonate (VP).
  • a phosphate or phosphate mimic at the 5’-end of the antisense strand e.g., a 5’-vinyl phosphonate (VP).
  • the 3’ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, 15 [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.
  • an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, pipe
  • the double stranded region may be 19-30 nucleotide pairs in length; 19-25 nucleotide pairs in length; 19-23 nucleotide pairs in length; 23-27 nucleotide pairs in length; or 21-23 nucleotide pairs in length.
  • each strand is independently no more than 30 nucleotides in length.
  • the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.
  • the dsRNA agent, or a pharmaceutically acceptable salt thereof comprises at least one single-stranded overhang, e.g., a single-stranded overhang that is 1, 2 or 3 25 nucleotides in length.
  • the dsRNA agent, or a pharmaceutically acceptable salt thereof is blunt-ended.
  • the dsRNA agent, or a pharmaceutically acceptable salt thereof further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
  • the phosphorothioate or methylphosphonate internucleotide linkage is at the 3’-terminus of one strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5’-terminus of one strand.
  • the phosphorothioate or methylphosphonate internucleotide linkage is at 35 both the 5’- and 3’-terminus of one strand.
  • the dsRNA agent, or a pharmaceutically acceptable salt thereof comprises 6-10, e.g., 6, 7, 8, 9, or 10, phosphorothioate or methylphosphonate internucleotide linkages. 4 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • the dsRNA agent, or a pharmaceutically acceptable salt thereof comprises 7 phosphorothioate or methylphosphonate internucleotide linkages.
  • the ⁇ v ⁇ 6 integrin targeting ligand the ⁇ v ⁇ 6 integrin targeting ligand comprises a structure represented by Formula (X): 5 or a salt thereof, wherein: or CH2; , wherein m is 0, 1, 2, 3, or 4; and each R 2 is independently R, or two R 2 groups on adjacent carbon atoms taken together with the atoms 10 to which they are bound form a fused 4 – 8 membered ring that is optionally substituted by 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group;
  • A is an 5-membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl;
  • 15 Q is -COOR1 or tetrazolyl (e.g., 1,2,3,4-tetrazol-5-yl), wherein R 1 is hydrogen or
  • the ⁇ v ⁇ 6 integrin targeting ligand of Formula (X) is 6 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO , wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 5
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group. In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group. In one embodiment, Y’ is O, R 1 is C1-6alkyl and R P is hydrogen. 25 In one embodiment, Y’ is S, R 1 is C1-6alkyl and R P is hydrogen. In one embodiment, Y’ is O, R 1 is C1-6alkyl and R P is a nitrogen protecting group. In one embodiment, Y’ is S, R 1 is C1-6alkyl and R P is a nitrogen protecting group. 7 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 56.
  • the ⁇ v ⁇ 6 integrin targeting ligand is wherein a broken bond represents a bond to the remainder of the dsRNA, or a pharmaceutically acceptable salt thereof.
  • the in vivo delivery enhancing moiety comprises at least one C10-C26 hydrocarbon chain. In one embodiment, the in vivo delivery enhancing moiety comprises at least one C 22 hydrocarbon chain. In one embodiment, the at least one C 22 hydrocarbon chains is an aliphatic, alicyclic, or 10 polyalicyclic compound.
  • the at least one C 22 hydrocarbon chains contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In one embodiment, the at least one C 22 hydrocarbon chains is a C 22 acid.
  • the C 22 acid is selected from the group consisting of docosanoic acid, 6- octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all- cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16- docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and cis-13-docosenoic acid.
  • the at least one C 22 hydrocarbon chains is a C 22 alcohol.
  • the C 22 alcohol is selected from the group consisting of 1-docosanol, 6- octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13-docosen-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, andcis-4,7,10,13,16,19-docosahexanol.
  • the at least one C 22 hydrocarbon chains is a C 22 amide.
  • the C 22 amide is selected from the group consisting of (E)-Docos-4- 25 enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Docos-11-enamide,12-Docosenamide, 8 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the at least one C10-C26 hydrocarbon chain is unsubstituted or 5 substituted with at least one functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.
  • the at least one C10-C26 hydrocarbon chains is substituted with a carboxylic acid group.
  • the in vivo delivery enhancing moiety is attached to the dsRNA agent, or 10 a pharmaceutically acceptable salt thereof, via a linker or via a carrier or via an internucleotide phosphate linkage. In one embodiment, the in vivo delivery enhancing moiety is attached to the dsRNA agent via a linker.
  • the linker comprises –(CH2)n-NH-C(O)- or –(CH2)n-NH-C(O)-(CH2)2- C(COOH)-NH-C(O)-, wherein n is a number from 1 to 20.
  • the linker comprises –(CH)2-O-(CH2CH2)-(O)-(CH2CH2)-NH-C(O)-.
  • the in vivo delivery enhancing moiety is represented by the following structure: 25 , 30 , 9 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the at least one ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the 3’ end antisense strand. In another embodiment, the at least one ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the 5’ end of the antisense strand. In one embodiment, the at least one ⁇ v ⁇ 6 integrin targeting ligand is conjugated to both the 5’-end and the 3’-end of the antisense strand. 10 In one embodiment, the at least one ⁇ v ⁇ 6 integrin targeting ligand is conjugated to an internal position of the antisense strand. In one embodiment, the at least one in vivo delivery enhancing moiety is conjugated to an internal position of the sense strand.
  • the at least one ⁇ v ⁇ 6 integrin targeting ligand and the at least one in vivo delivery enhancing moiety are both conjugated to the antisense strand. In one embodiment, the at least one ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the sense 35 strand and the at least one in vivo delivery enhancing moiety is conjugated to the antisense strand. In one embodiment, the at least one ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the antisense strand and the at least one in vivo delivery enhancing moiety is conjugated to the sense strand. 11 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the cell is a muscle cell, e.g., a skeletal muscle cell, a cardiac muscle cell, and/or a smooth muscle cell.
  • the cell is within a subject, e.g., a human, such as a subject having an DMPK-associated disorder, e.g., myotonic dystrophy type 1 (DM1).
  • contacting the cell with the dsRNA agent inhibits the expression of the 20 DMPK gene by at least 50%, 60%, 70%, 80%, 90%, or 95%.
  • inhibiting expression of the DMPK gene decreases DMPK protein level in serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.
  • the dsRNA agent is administered to the subject subcutaneously. 5 In another embodiment, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject intramuscularly. In one embodiment, the method further comprises administering to the subject an additional therapeutic agent for treatment of a DMPK-associated disorder.
  • the present disclosure further provides a kit, a vial, or a syringe comprising the dsRNA agent, 10 or a pharmaceutically acceptable salt thereof, of the disclosure or the pharmaceutical composition of the disclosure.
  • the present disclosure provides an RNA-induced silencing complex (RISC) comprising an antisense strand of any of the dsRNA agents, or a pharmaceutically acceptable salt thereof, of the disclosure.
  • RISC RNA-induced silencing complex
  • the iRNAs of the disclosure have been designed to target the human DMPK gene, including portions of the gene that are conserved in orthologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the specific target sites or the specific modifications in these iRNAs confer to the 35 iRNAs of the disclosure improved efficacy, stability, potency, durability, and safety. Accordingly, the present disclosure provides methods for treating and preventing a DMPK- associated disorder, e.g., myotonic dystrophy type 1 (DM1), using dsRNA compositions which effect 13 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • DM1 myotonic dystrophy type 1
  • the iRNAs of the disclosure include an RNA strand (the antisense strand) having a region which is up to about 30 nucleotides or less in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 5 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21- 30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a DMPK gene.
  • one or both of the strands of the double stranded RNA agents of the disclosure is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides 10 in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of a metabolic disorder-associated target gene, i.e., DMPK.
  • DMPK metabolic disorder-associated target gene
  • such dsRNA agents having longer length antisense strands may, for example, include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.
  • iRNAs of the disclosure enables the targeted degradation of the DMPK mRNAs in mammals. Using in vitro and in vivo assays, the present inventors have demonstrated that iRNAs targeting the gene can potently mediate RNAi, resulting in significant inhibition of expression of the DMPK gene.
  • compositions including these iRNAs are useful for treating a subject having a DMPK-associated disorder, e.g., myotonic dystrophy type 1 (DM1), or for treating a 20 subject at risk of developing a DMPK-associated disorder.
  • a DMPK-associated disorder e.g., myotonic dystrophy type 1 (DM1)
  • the present disclosure provides methods and combination therapies for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a DMPK gene, e.g., a DMPK-associated disorder, e.g., myotonic dystrophy type 1 (DM1), using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA 25 transcripts of a DMPK gene.
  • RISC RNA-induced silencing complex
  • the present disclosure also provides methods for preventing at least one symptom in a subject having a disorder that would benefit from inhibiting or reducing the expression of a DMPK gene, e.g., a DMPK-associated disorder, e.g., myotonic dystrophy type 1 (DM1).
  • a DMPK-associated disorder e.g., myotonic dystrophy type 1 (DM1).
  • the following detailed description discloses how to make and use compositions containing 30 iRNAs to inhibit the expression of DMPK, as well as compositions, uses, and methods for treating subjects that would benefit from inhibition and/or reduction of the expression of DMPK, e.g., subjects susceptible to or diagnosed with a DMPK-associated disorder, e.g., myotonic dystrophy type 1 (DM1). 35 I.
  • sense strand or antisense strand is 10 understood as “sense strand or antisense strand or sense strand and antisense strand.”
  • the term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%.
  • nucleotides in a nucleic acid molecule must be an integer.
  • at least 19 nucleotides of a 20 21 nucleotide nucleic acid molecule means that 19, 20, or 21 nucleotides have the indicated property.
  • methods of detection can include determination that the amount of analyte present is below the level of detection of the method. 30
  • the indicated sequence takes precedence.
  • the nucleotide sequence recited in the specification takes precedence.
  • “dystrophy myotonic protein kinase,” used interchangeably with the terms 35 “DMPK,” refers to the serine-threonine kinase that is closely related to other kinases that interact with members of the Rho family of small GTPases.
  • Substrates for this enzyme include myogenin, the beta- subunit of the L-type calcium channels, and phospholemman.
  • the 3' untranslated region of this gene contains 5-38 copies of a CTG trinucleotide repeat. Expansion of this unstable motif to 50-5,000 15 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO copies causes myotonic dystrophy type I, which increases in severity with increasing repeat element copy number. Repeat expansion is associated with condensation of local chromatin structure that disrupts the expression of genes in this region.
  • An exemplary sequence of a human DMPK mRNA transcript can be found at, for example, 5 GenBank Accession No.
  • NM_004409.5 (GI: 1677499213, SEQ ID NO:1; reverse complement, SEQ ID NO:2).
  • An exmplary sequence of mouse DMPK mRNA can be found at, for example, GenBank Accession No. NM_032418.3 (SEQ ID NO:3; reverse complement, SEQ ID NO:4).
  • An exemplary sequence of rat DMPK mRNA can be found at, for example, GenBank Accession No. NM_001415847 (SEQ ID NO:5; reverse complement, SEQ ID NO:6).
  • An exemplary sequence of 10 Macaca mulatta DMPK mRNA can be found at, for example, GenBank Accession No.
  • NM_001260568 (SEQ ID NO:7; reverse complement, SEQ ID NO:8).
  • target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a DMPK gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • the target portion of the sequence will be at least long enough to serve as a substrate for 30 RNAi-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a DMPK gene.
  • the target sequence is within the protein coding region of the DMPK gene. In another embodiment, the target sequence is within the 3’ UTR of the DMPK gene.
  • the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state. 35
  • the target sequence may be from about 19-36 nucleotides in length, e.g., about 19-30 nucleotides in length.
  • the target sequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20- 25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 16 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO nucleotides in length.
  • the target sequence is 19-23 nucleotides in length, optionally 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.
  • the term “strand comprising a sequence” refers to an oligonucleotide 5 comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. “G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively.
  • ribonucleotide or “nucleotide” can also refer to a modified nucleotide, as further detailed 10 below, or a surrogate replacement moiety (see, e.g., Table 1).
  • guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, 15 guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the disclosure by a nucleotide containing, for example, inosine.
  • adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the disclosure.
  • RNAi agent refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
  • RNAi RNA interference
  • the iRNA modulates, e.g., inhibits, the expression of a DMPK gene in a 25 cell, e.g., a muscle cell , within a subject, such as a mammalian subject.
  • RNAi agent and “dsRNA agent” may be used interchangeably herein.
  • an RNAi agent of the disclosure includes a single stranded RNA that interacts with a target RNA sequence, e.g., a DMPK mRNA sequence, to direct the cleavage of the 30 target RNA.
  • a target RNA sequence e.g., a DMPK mRNA sequence
  • Dicer Type III endonuclease
  • Dicer a ribonuclease-III-like enzyme, processes the dsRNA into 19- 23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363).
  • the siRNAs are then incorporated into an RNA-induced silencing complex 35 (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309).
  • RISC RNA-induced silencing complex 35
  • the disclosure relates to a 17 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the DMPK gene.
  • siRNA is also used herein to refer to an iRNA as described above.
  • the RNAi agent may be a single-stranded siRNA (ssRNAi) that is 5 introduced into a cell or organism to inhibit a target mRNA.
  • Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA.
  • the single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single- stranded siRNAs are described in U.S. Patent No.8,101,348 and in Lima et al., (2012) Cell 150:883- 894, the entire contents of each of which are hereby incorporated herein by reference.
  • an “iRNA” for use in the compositions, uses, and methods of the disclosure is a double stranded RNA and is referred to herein as a “double stranded RNA agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”.
  • dsRNA refers to a 15 complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a DMPK mRNA sequence.
  • a double stranded RNA dsRNA triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA 20 interference or RNAi.
  • each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide or a modified nucleotide.
  • an “iRNA” may include ribonucleotides with chemical modifications; an iRNA may 25 include substantial modifications at multiple nucleotides.
  • modified nucleotide refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or modified nucleobase, or any combination thereof.
  • modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases.
  • the modifications suitable for use in the 30 agents of the disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “iRNA” or “RNAi agent” for the purposes of this specification and claims.
  • the duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 19 to 36 base pairs in length, e.g., about 19-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 19-30, 19-29, 18 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also 5 contemplated to be part of the disclosure.
  • the two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting 10 RNA chain is referred to as a “hairpin loop.”
  • a hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides.
  • the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin 15 loop can be 4-8 nucleotides.
  • the two strands of double-stranded oligomeric compound can be linked together. The two strands can be linked to each other at both ends, or at one end only. By linking at one end is meant that 5'-end of first strand is linked to the 3'-end of the second strand or 3'- end of first strand is linked to 5'-end of the second strand.
  • first strand is linked to 3'-end of second strand and 3'-end of first strand is linked to 5'-end of second strand.
  • the two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiemtns, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10.
  • the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, 25 wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide.
  • N is a modified or unmodified nucleotide
  • R is a modified or unmodified purine nucleotide.
  • Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs.
  • the duplex region can be equal to or less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
  • the hairpin oligomeric compounds can have a single strand overhang or terminal unpaired 35 region, in some embodiments at the 3', and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more generally 2-3 nucleotides in length.
  • the hairpin oligomeric compounds that can induce RNA interference are also referred to as "shRNA" herein.
  • shRNA The hairpin oligomeric compounds that can induce RNA interference
  • 19 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not be, but can be covalently connected.
  • the connecting 5 structure is referred to as a “linker.”
  • the RNA strands may have the same or a different number of nucleotides.
  • an RNAi may comprise one or more nucleotide overhangs.
  • at least one strand comprises a 3’ overhang of at least 1 nucleotide.
  • at least one 10 strand comprises a 3’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides.
  • at least one strand of the RNAi agent comprises a 5’ overhang of at least 1 nucleotide.
  • At least one strand comprises a 5’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides.
  • both the 3’ and the 5’ end of one strand of the RNAi agent comprise an overhang of at least 1 15 nucleotide.
  • an iRNA agent of the disclosure is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a DMPK mRNA sequence, to direct cleavage of the target RNA.
  • an iRNA of the disclosure is a dsRNA of 24-30 nucleotides that 20 interacts with a target RNA sequence, e.g., a DMPK mRNA sequence, to direct the cleavage of the target RNA.
  • a target RNA sequence e.g., a DMPK mRNA sequence
  • nucleotide overhang refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double stranded iRNA. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, there is a nucleotide 25 overhang.
  • a dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand, or any combination thereof.
  • the nucleotide(s) of an overhang can be 30 present on the 5'-end, 3'-end, or both ends of either an antisense or sense strand of a dsRNA.
  • the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-end or the 5’-end.
  • the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-end or the 5’-end.
  • one or more of the nucleotides in the overhang is 35 replaced with a nucleoside thiophosphate.
  • the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-end or the 5’- end.
  • the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 20 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the antisense strand of a dsRNA has a 1-10 nucleotides, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3’-end or the 5’-end.
  • the 5 overhang on the sense strand or the antisense strand, or both can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length.
  • an extended overhang is on the sense strand of the duplex.
  • an extended overhang is present on the 3’ end of the sense strand of the duplex.
  • an extended overhang is present on the 5’ end 10 of the sense strand of the duplex.
  • an extended overhang is on the antisense strand of the duplex.
  • an extended overhang is present on the 3’end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5’end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the extended overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang 15 includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNA agent, i.e., no nucleotide overhang.
  • a “blunt ended” double stranded RNA agent is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
  • the 20 RNAi agents of the disclosure include RNAi agents with no nucleotide overhang at one end (i.e., agents with one overhang and one blunt end) or with no nucleotide overhangs at either end. Most often such a molecule will be double-stranded over its entire length.
  • antisense strand or "guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a DMPK 25 mRNA.
  • region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a DMPK nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the 30 molecule.
  • a double stranded RNA agent of the disclosure includes a nucleotide mismatch in the antisense strand.
  • the antisense strand of the double stranded RNA agent of the disclosure includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with 35 the target mRNA.
  • the antisense strand double stranded RNA agent of the disclosure includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand.
  • a double stranded RNA agent of the disclosure includes a nucleotide mismatch in the sense strand.
  • the sense strand of the double stranded RNA agent of the disclosure includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand.
  • the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3’-end of the iRNA.
  • the nucleotide mismatch is, for 5 example, in the 3’-terminal nucleotide of the iRNA agent.
  • the mismatch(s) is not in the seed region.
  • an RNAi agent as described herein can contain one or more mismatches to the target sequence.
  • an RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein 10 contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5’- or 3’-end of the region of complementarity.
  • RNAi agent for 15 a 23 nucleotide RNAi agent, the strand which is complementary to a region of DMPK, generally does not contain any mismatch within the central 13 nucleotides.
  • the methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a target gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a DMPK gene is important, especially if the 20 particular region of complementarity in the target gene is known to have polymorphic sequence variation within the population.
  • sense strand or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein. 25 As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.
  • cleavage region refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs.
  • the cleavage region comprises three bases on either end of, and immediately 30 adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.
  • the term “complementary,” when used to 35 describe a first nucleotide sequence in relation to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for 22 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO example be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 o C or 70 o C for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an 5 organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • Complementary sequences within an iRNA include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an 10 oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences.
  • Such sequences can be referred to as “fully complementary” with respect to each other herein.
  • first sequence is referred to as “substantially complementary” with respect to a second sequence herein
  • the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched 15 base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression, in vitro or in vivo.
  • two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity.
  • a dsRNA comprising one oligonucleotide 20 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.
  • “Complementary” sequences, as used herein, can also include, or be formed entirely from, non- Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as 25 the above requirements with respect to their ability to hybridize are fulfilled.
  • non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairing.
  • the terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between two oligonucletoides or polynucleotides, such as the antisense strand of a 30 double stranded RNA agent and a target sequence, as will be understood from the context of their use.
  • a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding DMPK).
  • mRNA messenger RNA
  • a polynucleotide is complementary to at least a part of a DMPK mRNA if the sequence is substantially 35 complementary to a non-interrupted portion of an mRNA encoding DMPK.
  • the antisense strand polynucleotides disclosed herein are fully complementary to the target gene, i.e., DMPK sequence.
  • the antisense strand polynucleotides disclosed herein are substantially complementary to the target gene sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 1, 3, 5, and 7, for DMPK, or a fragment of SEQ ID NOs: 1, 3, 5, and 7, 5 such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.
  • the antisense polynucleotides disclosed herein are substantially complementary to the target DMPK sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide 10 sequences in any one of Tables 2-3, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-3, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.
  • an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target 15 DMPK sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 2, 4, 6, and 8, or a fragment of any one of SEQ ID NOs: 2, 4, 6, and 8, 10, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.
  • an iRNA of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target DMPK sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of any one of Tables 2-3, or a fragment of any one of the antisense 25 strand nucleotide sequences in any one of Tables 2-3, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.
  • the double-stranded region of a double-stranded iRNA agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.
  • the antisense strand of a double-stranded iRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the sense strand of a double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
  • the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length. In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length. 24 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.
  • the sense strand of the iRNA agent is 21-nucleotides in length
  • the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 5 consecutive base pairs having a 2-nucleotide long single stranded overhang at the 3'-end.
  • an “iRNA” includes ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a dsRNA molecule, are encompassed by “iRNA” for the purposes of this specification and claims.
  • an agent for use in the methods and compositions of the disclosure is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism.
  • the single-stranded antisense oligonucleotide molecule is 15 complementary to a sequence within the target mRNA.
  • the single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347- 355.
  • the single-stranded antisense oligonucleotide molecule may be about 14 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence.
  • the single- 20 stranded antisense oligonucleotide molecule may comprise a sequence that is at least about 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting 25 ligand, and (iv) 2’-fluoro modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2’-O- methyl modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2’-fluoro modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5’ end); and (iii) phosphorothioate linkages between 30 nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (count
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting 35 ligand; (iv) 2’-fluoro modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2’-O- methyl modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5’ end); and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a 25 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting 10 ligand; (iv) 2’-O-methyl modifications at positions 1 to 6, 8, 10, and 12 to 21, 2’-fluoro modifications at positions 7 and 9, and a deoxy-nucleotide (e.g.
  • dT phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 15 to 23, and 2’-fluoro modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5’ end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agent has a two- nucleotide overhang at the 3’-end of the antisense strand, and a
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting ligand; (iv) 2’-O-methyl modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2’-fluoro modifications at positions 7, 9, 11, 13, and 15 (counting from the 5’ end); and (v) phosphorothioate 25 internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’- O-methyl modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2’-fluoro modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5’
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting 35 ligand; (iv) 2’-O-methyl modifications at positions 1 to 9, and 12 to 21, and 2’-fluoro modifications at positions 10, and 11 (counting from the 5’ end); and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications 26 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting ligand; (iv) 2’-fluoro modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2’-O-methyl 10 modifications at positions 2, 4, 6, 8, 12, and 14 to 21 (counting from the 5’ end); and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2’-fluoro modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from 15 the
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting ligand; (iv) 2’-O-methyl modifications at positions 1 to 6, 8, and 12 to 21, and 2’-fluoro modifications at positions 7, and 9 to 11 (counting from the 5’ end); and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting 25 from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2’-fluoro modifications at positions 2, 6, 9, 14, and 16 (counting from the 5’ end); and (iii)
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting ligand; (iv) 2’-O-methyl modifications at positions 1 to 6, 8, and 12 to 21, and 2’-fluoro modifications 35 at positions 7, and 9 to 11; and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2’-fluoro modifications at positions 2, 6, 8, 9, 14, and 16 27 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Aln
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 19 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting ligand; (iv) 2’-O-methyl modifications at positions 1 to 4, 6, and 10 to 19, and 2’-fluoro modifications at positions 5, and 7 to 9; and (v) phosphorothioate internucleotide linkages between nucleotide 10 positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 21 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2’-fluoro modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5’ end); and (iii) phosphorothioate internu
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting 20 ligand; (iv) 2’-O-methyl modifications at positions 1 to 8, and 12 to 21, and 2’-fluoro modifications at positions 9 to 11; and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3, 4, 6, 8 to 11, 13, 15 to 23, and a 2’-fluoro modification at position 14 (counting from the 5’ end), and 2’- 25 deoxy-modified nucleotides at positions 2, 5, 7, and 12; and
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety, (iii) an ⁇ v ⁇ 6 integrin targeting ligand; (iv) 2’-O-methyl modifications at positions 1 to 6, 8, and 12 to 21, and 2’-fluoro modifications at positions 7, and 9 to 11; and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and (b) an 35 antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3 to 4, 6, 8 to 13, 15, and 17 to 23, 2’-fluoro modifications at positions 2, 14, and 16, a 2’-deoxy- modified nucleotide at position 5, and a GNA at position 7 (count
  • the dsRNA agent has a two-nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: 5 (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety at position 5, 6, 16 or 17 (counting from the 5’ end), (iii) an ⁇ v ⁇ 6 integrin targeting ligand on the 3’ end; (iv) 2’-O-methyl modifications at positions 1 to 6, 8, and 12 to 21, and 2’-fluoro modifications at positions 7 and 9 to 11 (counting from the 5’ end); and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between positions 20 and 21, and between 10 position 21 and the ⁇ v ⁇ 6 integrin targeting ligand (counting from the 5’ end); and (b) an antisense strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3 to
  • a dsRNA agent of the disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) an in vivo delivery enhancing moiety at position 5, 6, 16 or 17 20 (counting from the 5’ end), (iii) an ⁇ v ⁇ 6 integrin targeting ligand on the 3’ end; (iv) 2’-O-methyl modifications at positions 1 to 6, 8, and 12 to 21, and 2’-fluoro modifications at positions 7, and 9 to 11 (counting from the 5’ end); and (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between positions 20 and 21, and between position 21 and the ⁇ v ⁇ 6 integrin targeting ligand (counting from the 5’ end); and (b) an antisense 25 strand having: (i) a length of 23 nucleotides; (ii) 2’-O-methyl modifications at positions 1, 3 to
  • the sense strand of the dsRNA agent has 2’-F modifications at positions 7, 9, 10 and 11 (counting from the 5’ end). In some embodiments, the sense strand of the dsRNA agent has 2’-F modifications at positions 9 to 11 (counting from the 5’ end). In some 35 embodiments, the sense strand of the dsRNA agent has 2’-F modifications at positions 7 to 9 (counting from the 5’ end). In some embodiments, the sense strand of the dsRNA agent has 2’-F modifications at positions 5, 7, 8, and 9 (counting from the 5’ end).
  • the sense strand of the dsRNA agent has 2’-F modifications at positions 9, 11, and 13 (counting from the 5’ 29 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO end). In some embodiments, the sense strand of the dsRNA agent has 2’-F modifications at positions 9, 11, and 12 (counting from the 5’ end). In some embodiments, the sense strand of the dsRNA agent has 2’-F modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5’ end).
  • the antisense strand of the dsRNA agent has 2’-F modifications at 5 positions 1, 2, 6, 14, and 16 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent has 2’-F modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent has 2’-F modifications at positions 2, 4, 6, 12, 14, 16, 18, and 19 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent has 2’-F modifications at positions 2, 3, 5, 10, 10 12, 14, and 16 (counting from the 5’ end).
  • the antisense strand of the dsRNA agent has 2’-F modifications at positions 2, 4, 12, 14 and 16 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent has 2’-F modifications at positions 2, 12, 14, and 16 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent has 2’-F modifications at positions 2, 6, 14, and 16 (counting from the 5’ end). In some embodiments, the 15 antisense strand of the dsRNA agent has 2’-F modifications at positions 2, 14, and 16 (counting from the 5’ end).
  • the antisense strand of the dsRNA agent has 2’-F modifications at positions 2 and 14 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent has a 2’-F modification at only position 14 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent has 2’-deoxy-modified 20 nucleotides at positions 2, 5, 7, and 12 (counting from the 5’ end). In some embodiments, the antisense strand of the dsRNA agent comprises a 2’-deoxy-modified nucleotide at position 5 (counting from the 5’ end).
  • the antisense strand of the dsRNA agent comprises a GNA modified nucleotide at position 7 (counting from the 5’ end).
  • a “pharmaceutically acceptable salt” of the dsRNA agents of the disclosure includes any salt which is pharmaceutically acceptable, e.g., a sodium salt of the dsRNA agent.
  • an “alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand”, as used herein, includes any moiety (e.g., peptides and small molecules) which bind an ⁇ v ⁇ 6 integrin and are able to mediate delivery of a dsRNA agent to which they are attached to skeletal muscle (e.g., a skeletal muscle cell or skeletal 30 muscle tissue) and/or cardiac muscle (e.g., a cardiac muscle cell or cardiac muscle tissue).
  • the ⁇ v ⁇ 6 integrin targeting ligands bind ⁇ v ⁇ 6 integrin or the ⁇ v ⁇ 6 integrin receptor on skeletal muscle/cardiac myocytes (cells).
  • Exemplary ⁇ v ⁇ 6 integrin targeting ligands are described below. Exemplary ⁇ v ⁇ 6 integrin targeting ligands are also described in WO2024/086633, the entire contents of which are incorporated by reference herein. 35
  • the phrase “contacting a cell with an iRNA,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly.
  • the iRNA may be put into physical contact with the cell by the 30 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO individual performing the method, or alternatively, the iRNA may be put into a situation that will permit or cause it to subsequently come into contact with the cell.
  • Contacting a cell in vitro may be done, for example, by incubating the cell with the iRNA.
  • Contacting a cell in vivo may be done, for example, by injecting the iRNA into or near the tissue 5 where the cell is located, or by injecting the iRNA into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located.
  • the iRNA may contain or be coupled to alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand that mediates delivery to muscle tissue and at least one in vivo delivery enhancing moiety. 10
  • Combinations of in vitro and in vivo methods of contacting are also possible.
  • a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.
  • contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell.
  • Absorption or uptake of an iRNA can occur through unaided diffusion or active cellular processes, or by auxiliary 15 agents or devices.
  • Introducing an iRNA into a cell may be in vitro or in vivo.
  • iRNA can be injected into a tissue site or administered systemically.
  • In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
  • lipid nanoparticle is a vesicle comprising a lipid layer encapsulating a 20 pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed.
  • LNPs are described in, for example, U.S. Patent Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.
  • a “subject” is an animal, such as a mammal, including a primate (such as a 25 human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously.
  • a primate such as a 25 human, a non-human primate, e.g., a monkey, and a chimpanzee
  • a non-primate such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse
  • the subject is a human, such as a human being treated or assessed for a disease or disorder that would benefit from reduction in DMPK expression; a human at risk for a disease or disorder that would benefit from 30 reduction in DMPK expression; a human having a disease or disorder that would benefit from reduction in DMPK expression; or human being treated for a disease or disorder that would benefit from reduction in DMPK expression as described herein.
  • the subject is a female human.
  • the subject is a male human.
  • the subject is an adult subject.
  • the subject is a pediatric subject.
  • treating refers to a beneficial or desired result, such as reducing at least one sign or symptom associated with DMPK expression or DMPK protein production, e.g., a DMPK-associated disease, e.g., myotonic dystrophy type 1 (DM1), in a subject.
  • Treatment also includes a reduction of one or more sign or symptoms associated with unwanted 31 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO DMPK expression; diminishing the extent of unwanted DMPK activation or stabilization; amelioration or palliation of unwanted DMPK activation or stabilization.
  • “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.
  • the term “lower” in the context of the level of DMPK in a subject or a disease marker or 5 symptom refers to a statistically significant decrease in such level.
  • the decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more.
  • a decrease is at least 20%.
  • the decrease is at least 50% in a disease marker, e.g., protein or gene expression level.
  • “Lower” in the context of the level of DMPK in a subject is a decrease to a level accepted as within the range of 10 normal for an individual without such disorder.
  • “lower” is the decrease in the difference between the level of a marker or symptom for a subject suffering from a disease and a level accepted within the range of normal for an individual.
  • the term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from a DMPK- 15 associated disorder towards or to a level in a normal subject not suffering from a DMPK-associated disorder.
  • prevention when used in reference to a disease, disorder or 20 condition thereof, may be treated or ameliorated by a reduction in expression of a DMPK gene or production of a DMPK protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of a DMPK- associated disorder, e.g., DM1.
  • DMPK-associated disorder or “DMPK-associated disease” is a disease or disorder that would benefit from reduction in the mRNA expression or activity of DMPK.
  • DMPK-associated disease is a disease or disorder that is caused by, or associated with, 30 DMPK mRNA expression or DMPK protein production.
  • DMPK-associated disease includes a disease, disorder or condition that would benefit from a decrease in DMPK mRNA expression or DMPK protein activity.
  • the DMPK-associated disease is a myotonic dystrophy.
  • Myotonic dystrophy refers to a part of a group of inherited disorders called 35 muscular dystrophies. It is the most common form of muscular dystrophy that begins in adulthood. Myotonic dystrophy is characterized by progressive muscle wasting and weakness. People with this disorder often have prolonged muscle contractions (myotonia) and are not able to relax certain muscles after use.
  • myotonic dystrophy Other signs and symptoms of myotonic dystrophy include clouding of the lens of 32 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO the eye (cataracts) and abnormalities of the electrical signals that control the heartbeat (cardiac conduction defects).
  • Some affected individuals develop a condition called diabetes mellitus, in which blood sugar levels can become dangerously high.
  • the features of myotonic dystrophy often develop during a person's twenties or thirties, although they can occur at any age.
  • the severity of the condition 5 varies widely among affected people, even among members of the same family. There are two major types of myotonic dystrophy: type 1 and type 2.
  • type 2 tends to be milder than type 1.
  • the muscle weakness associated with type 1 particularly affects muscles farthest from the center of the body (distal muscles), such as those of the lower legs, hands, neck, and face.
  • Muscle weakness in type 2 primarily involves muscles close to the center of the body 10 (proximal muscles), such as the those of the neck, shoulders, elbows, and hips.
  • the two types of myotonic dystrophy are caused by mutations in different genes.
  • Myotonic dystrophy type 1 is caused by mutations in the DMPK gene, while type 2 results from mutations in the CNBP gene.
  • the protein produced from the DMPK gene likely plays a role in communication within cells.
  • the protein produced from the CNBP gene is found primarily in the heart and in skeletal muscles, where it helps regulate the function of other genes. Similar changes in the structure of the DMPK and CNBP genes cause myotonic dystrophy type 1 and type 2. In each case, a segment of DNA is abnormally repeated many times, forming an unstable region in the gene. The gene with the abnormal segment produces an unusually 20 long messenger RNA, which is a molecular blueprint of the gene that guides the production of proteins. The unusually long messenger RNA forms clumps inside the cell that interfere with the production of many other proteins.
  • the DMPK-associated disease is myotonic dystrophy type 1 (DM1).
  • “Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a DMPK-associated disorder, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease 30 or one or more symptoms of disease).
  • the "therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated. “Prophylactically effective amount,” as used herein, is intended to include the amount of an 35 RNAi agent that, when administered to a subject having a DMPK-associated disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease.
  • the “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is 33 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
  • a “therapeutically-effective amount” or “prophylactically effective amount” also includes an 5 amount of an RNAi agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any treatment.
  • the iRNA employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, 10 materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically- 15 acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing aid e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the 20 subject being treated.
  • Pharmaceutically acceptable carriers include carriers for administration by injection.
  • sample includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.
  • biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, 25 lymph, urine, saliva, and the like.
  • Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes).
  • a “sample derived from a subject” refers to urine obtained from 30 the subject.
  • a “sample derived from a subject” can refer to blood or blood derived serum or plasma from the subject.
  • alkyl group can be both a monovalent radical or divalent radical; in the latter case, it would be apparent to one skilled in the art that an additional hydrogen atom is removed from a monovalent alkyl radical to provide a suitable divalent moiety.
  • oligonucleotides may be an RNA, a DNA, a single-stranded RNA, such as an 10 antisense oligonucleotide (ASO), a sense or an antisense strand of an siRNA, and oligonucleotide derivatives such as phosphorodiamidate morpholino oligomers (PMOs).
  • ASO antisense oligonucleotide
  • siRNA oligonucleotide derivatives
  • PMOs phosphorodiamidate morpholino oligomers
  • conjugated means that the moiety is covalently attached to a dsRNA agent directly or via a linker or via a carrier or via an internucleotide phosphate linkage.
  • alkenyl as used herein, means a straight or branched chain hydrocarbon containing from 2 to 10 carbons, unless otherwise specified, and containing at least one carbon-carbon double 20 bond.
  • Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2- methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and 3,7-dimethylocta-2,6-dienyl.
  • alkynyl as used herein means a straight or branched hydrocarbon chain containing from 2 to 10 carbons, unless otherwise specified, and containing at least one carbon-carbon triple 25 bond. Representative examples of alkynyl include, but are not limited to, 1-butynyl, 2-butynyl, 1- propynyl and the like.
  • alkoxy as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
  • alkoxy examples include, but are not limited to, methoxy, ethoxy, n-propoxy, 2-propoxy, n-butoxy, tert-butoxy, n-pentyloxy, and n- 30 hexyloxy.
  • alkyl as used herein, means a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms, unless otherwise specified.
  • alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec -butyl, iso-butyl, tert-butyl, n- pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n- 35 heptyl, n-octyl, n-nonyl, and n-decyl.
  • aryl means a phenyl (i.e., monocyclic aryl); naphthyl or azulenyl; a bicyclic ring system containing a phenyl fused to a cycloalkyl, cycloalkenyl, or heterocyclyl ring.
  • bicyclic aryls include, but are not limited to, azulenyl, naphthyl, 2,3- dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2,3-dihydroinden-3-yl, 2,3-dihydroinden-4-yl, 2,3- 5 dihydroinden-5-yl, 2,3-dihydroindol-1-yl, indolin-2-yl, indolin-3-yl, indolin-4-yl, indolin-5-yl, indolin-6-yl, indolin-7-yl, inden-1-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen-1-yl, 5,6,7,8- tetrahydronaphthalen-1-
  • the bicyclic aryl is (i) naphthyl or (ii) a phenyl ring fused to either a 5 25 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • arylalkyl means an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined 30 herein.
  • Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3- phenylpropyl, and 2-naphth-2-ylethyl.
  • azido means a -N3 group.
  • carboxy means a -COOH group.
  • cyano and "nitrile” as used herein, mean a -CN group.
  • cycloalkyl as used herein, means a monocyclic or a bicyclic cycloalkyl ring system.
  • Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 10 carbon atoms, where such groups are saturated. Examples of monocyclic cycloalkyls include cyclopropyl, 36 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fused bicyclic rings.
  • Bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non-adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form - 5 (CH2)w-, where w is 1, 2, or 3).
  • Representative examples of bridged bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane.
  • Fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to a monocyclic cycloalkyl.
  • Representative examples of fused bicyclic ring systems include, but are not limited to, decaliyl.
  • Cycloalkyl groups 10 are optionally substituted with one or two groups which are independently oxo or thia.
  • the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to a 5 or 6 membered monocyclic cycloalkyl, wherein the fused bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia.
  • Cycloalkenyl refers to a monocyclic or a bicyclic cycloalkenyl ring system. 15 Monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon-carbon double bond), but not aromatic. Examples of monocyclic ring systems include cyclopentenyl and cyclohexenyl. Bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings.
  • Bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non-adjacent carbon atoms of the monocyclic ring 20 are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form -(CH2)w-, where w is 1, 2, or 3).
  • alkylene bridge of between one and three additional carbon atoms
  • bicyclic cycloalkenyls include, but are not limited to, norbornenyl and bicyclo[2.2.2]oct-2-enyl.
  • Fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a monocyclic cycloalkyl or a monocyclic cycloalkenyl. Cycloalkenyl groups are optionally substituted with one or two groups 25 which are independently oxo or thia.
  • the term “monocyclic ring”, as used herein, comprises monocyclic aryl, monocyclic cycloalkyl, monocyclic cycloalkenyl and monocyclic heterocyclyl.
  • halo or "halogen” as used herein, means -CI, -Br, -I or -F.
  • H means hydrogen.
  • haloalkyl means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.
  • heteroaryl as used herein, means a monocyclic heteroaryl or a bicyclic ring 35 system containing at least one heteroaromatic ring (i.e., a monocyclic or bicyclic aromatic ring system containing at least one heteroatom within the aromatic system).
  • the monocyclic heteroaryl can be a 5 or 6 membered ring.
  • the 5 membered ring consists of two double bonds and one, two, three or four nitrogen atoms and optionally one oxygen or sulfur atom.
  • the 6 membered ring consists of three 37 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO double bonds and one, two, three or four nitrogen atoms.
  • the 5 or 6 membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl.
  • monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, 5 pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl.
  • the bicyclic heteroaryl consists of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups which are independently oxo or thia.
  • bicyclic 10 heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, benzothiazolyl, cinnolinyl, 5 ,6-dihydroquinolin-2-yl, 5,6- dihydroquinolin-8-yl, 5,6-dihydroisoquinolin-1-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl, 5,6,7,8-tetrahydroquinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroquinolin-5-yl, 5,6,7,8-tetrahydro
  • the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic 20 heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • heteroarylalkyl and "-alkylheteroaryl” as used herein, means a heteroaryl, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein.
  • Representative examples of heteroarylalkyl include, but are not limited to, fur-3-ylmethyl, 1H- imidazol-2-ylmethylm 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl, pyridin-3-ylmethyl, pyridin-4- ylmethyl, pyrimidin-5-ylmethyl, 2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.
  • heterocyclyl as used herein, means a monocyclic heterocycle or a bicyclic heterocycle.
  • the monocyclic heterocycle is a 3, 4, 5, 6, or 7 membered ring containing at least one 30 heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
  • the 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S.
  • the 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the 6 or 7 membered ring contains zero, one, or two double bonds and one, two or three heteroatoms selected 35 from the group consisting of O, N and S.
  • monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxan-2-yl, 1,3- dioxolan-2-yl, 1,3-dithiolan-2-yl, 1,2-dithiolan-3-yl, 1,2-dithiolan-4-yl, 1,3-dithian-2-yl, 1,2-dithian-3- yl, 1,2-dithian-4-yl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, 38 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1- dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.
  • the bicyclic 5 heterocycle is a monocyclic heterocycle fused to either a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocycle.
  • Representative examples of bicyclic heterocyclyls include, but are not limited to, decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, and octahydrobenzofuranyl.
  • Heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the bicyclic heterocyclyl is a 5 or 6 10 membered monocyclic heterocyclyl ring fused to a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5 or 6 membered monocyclic heterocyclyl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.
  • hydroxy or “hydroxyl” as used herein means an -OH group.
  • thiol as 15 used herein means an –SH group.
  • hydroxyl protecting group refers to a labile chemical moiety which protects a hydroxyl group against undesired reactions during synthetic procedure(s). After the synthetic procedure(s), the hydroxy protecting group may be selectively removed. Hydroxy protecting groups as known in the art are described generally in T. H. Greene and P. G. M. Wuts, Protective 20 Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999).
  • hydroxyl protecting groups include, but are not limited to, benzyloxycarbonyl, 4- nitrobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, methoxycarbonyl, tert-butoxycarbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl, 2,2,2- trichloroethoxycarbonyl, 2-(trimethylsilyl) ethoxycarbonyl, 2-furfuryloxycarbonyl, allyloxycarbonyl, 25 acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, l,l-dimethyl-2-propenyl, 3-methyl-3-butenyl, allyl, ally
  • hydroxyl protecting groups can be selected from acetyl (Ac or — C(O)CH3), benzoyl (Bz or —C(O)C6H5), and trimethylsilyl (TMS or -Si(CH3)3).
  • nitro as used herein means a -NO2 group.
  • saturated as used herein means the referenced chemical structure does not contain 35 any multiple carbon-carbon bonds. For example, a saturated cycloalkyl group as defined herein includes cyclohexyl, cyclopropyl, and the like.
  • amine or “amino” encompasses compounds where a nitrogen atom is covalently bonded to at least one carbon or heteroatom.
  • alkyl amino includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
  • dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups. 5
  • unsaturated as used herein means the referenced chemical structure contains at least one multiple carbon-carbon bond (i.e., double or triple bond, or both), but is not aromatic.
  • an unsaturated cycloalkyl group as defined herein includes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.
  • the term “leaving group” as used herein means an atom or group (charged or uncharged) that 10 becomes detached from an atom in what is considered to be the residual or main part of the substrate in a specified reaction.
  • the specified reaction herein unless otherwise noted, is an SN1 or an SN2 reaction as is understood by one skilled in the art. In certain embodimentns, the specified reaction herein, is an SN2 reaction.
  • a“substituted” as used herein, whether preceded by the term“optionally” or not, 15 means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • a“substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in 20 any given structure is substituted, the substituent is either the same or different at each position.
  • Suitable substituents include, but are not limited to, halogen, hydroxy, thiol, nitro, alkoxy, azido, carboxy, cyano, amino, C1-6alkyl, C2-6alkenyl, C1-6alkoxy, C1-6alkylamino.
  • support linking group means a divalent chemical moiety that covalent connects a surface-bound functional group of a solid support (e.g., an amino group of an amino-modified solid support) to 25 another chemical moiety.
  • reactive pair means two functional groups known by one skilled in the art to be capable of reacting, alone or in the presence of other reagents, to form a covalent linkage between the two chemical entities which each contain one of member of the reactive pair, respectively; the latter, herein, is referred to as “linking group formed by a reactive pair”.
  • a reactive pair is a click pair, i.e., two functional groups capable of reacting in a click reaction to form a covalent linkage.
  • the term “Michael acceptor” as used herein means an alpha, beta-unsaturated compound capable of reacting with a nucleophile at the beta-carbon of the electrophilic alkene of the alpha, beta -unsaturated compound.
  • alpha, beta-unsaturated compound include, but are not limited 35 to, such as an alpha, beta-unsaturated aldehyde, ester, amide, sulfonyl, ketone, nitrile, or nitro.
  • Alpha, beta-unsaturated refers to the carbon-carbon multiple bond that connects the carbon atom that is immediately adjacent to the referenced aldehyde, ester, amide, sulfonyl, ketone, nitrile, or nitro group, to its adjacent carbon atom.
  • Michael acceptor group include, but are not limited 40 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO to, N-maleimide, acrylaldehyde, acrylonitrile, acrylic acid, acrylamide (e.g., N-isoproprylacrylamide), acrylate esters (e.g., methyl acrylate), vinyl sulfones, vinylsulfonates, and vinylsulfonamides.
  • nitrogen protecting group means those functional groups that are well known in the art and include those described in detail, for example, in Protecting Groups in 5 Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference.
  • Suitable nitrogen protecting groups include but are not limited to, allyl (Alloc), acetyl (Ac), Adpoc (1-(1-Adamantyl)-1-methylethoxycarbonyl), Boc ( tert-butyloxy carbonyl), Dde Dnp (2,4-dinitrophenyl), Mmt (4-methoxytrityl), Mtt (4-methyltrityl), Teoc (2-trimethylsilylethoxycarbonyl, Tfa (trifluoroacetyl),10 optionally substituted trityl (e.g., trityl (Trt), 2-chlorotrityl (Clt), 4-methoxytrityl (Mmt), 4- methyltrityl (Mtt), 4,4’-dimethoxytrityl (DMT)), and 4,4’,4’’-trimethoxytrityl), optionally substituted benzyloxycarbonyl (e.g., benz
  • the term “remainder of the dsRNA agent”, refers to an oligonucleotide strand (i.e. the sense strand or the antisense strand) that forms a dsRNA agent and any intervening linking group that connects the parent moiety (e.g., one or more ⁇ v ⁇ 6 integrin targeting ligand or one or more 25 in vivo delivery enhancing moieties) to the oligonucleotide strand.
  • linker refers to a chemical moiety connecting at least one ⁇ v ⁇ 6 integrin targeting ligand or at least one in vivo delivery enhancing moiety to at least one strand of the dsRNA agent of the disclosure.
  • iRNAs of the Disclosure Described herein are iRNAs for use in the methods of the present disclosure.
  • the iRNAs are double stranded ribonucleic acid (dsRNA) molecules.
  • the dsRNA agent targets an DMPK gene and inhibits the expression of the DMPK gene.
  • the iRNA agent 41 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO includes dsRNA molecules for inhibiting the expression of an DMPK gene in a cell, such as a liver cell, such as a muscle cell within a subject, e.g., a mammal, such as a human having DM1.
  • the iRNA agents provided herein comprise a sense strand and an antisense strand and at least one of the strands is modified for targeting delivery to extrahepatic tissue, e.g., muscle tissue, e.g., 5 skeletal muscle tissue and/or cardiac muscle tissue.
  • the iRNA agents are modified by conjugation to at least one alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin ligand and conjugation to at least one in vivo delivery enhancing moiety.
  • integrin ligand is meant any ligand that binds to an integrin or an integrin receptor. Integrin ligands comprise binding sequences that are recognized and bound by integrins or ntegrin 10 receptors.
  • Various ligands, including peptides and small molecules, which bind ⁇ v ⁇ 6 integrins or ⁇ v ⁇ 6 integrin receptors ( ⁇ v ⁇ 6 integrin targeting ligands) have been designed and synthesized herein and in, e.g., U.S.
  • An exemplary linker to conjugate a ⁇ v ⁇ 6 integrin targeting ligand to the dsRNA agent is: 15 42 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5 43 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO .
  • at least one of the strands of the iRNA agent is conjugated to at least one ⁇ v ⁇ 6 integrin targeting ligand and at least one in vivo delivery enhancing moiety.
  • the 5 iRNA agent may be conjugated to one, two, three, four, or more ⁇ v ⁇ 6 integrin targeting ligands and one, two, three, four or more in vivo delivery enhancing moieties.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to an extrernal position of sense strand, e.g., the 3’-end of the sense strand, to the 5’-end of the sense strand, or to both the 5-end and the 3’-end of the 10 sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to an internal position of the sense strand. In other embodiments, the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the antisense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to an external position of the antisense strand, e.g., the 3’-end of the antisense strand, to the 5’-end of the antisense strand, or to both the 5-end and 15 the 3’-end of the antisense strand. In some embodiments, the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to an internal position of the antisense strand.
  • the in vivo delivery enhancing moiety is conjugated to the sense strand.
  • the in vivo delivery enhancing moiety may be conjugated to an extrernal position of sense strand, e.g., the 3’-end of the sense strand, to the 5’-end of the sense strand, or to both the 5-end and 20 the 3’-end of the sense strand.
  • the in vivo delivery enhancing moiety may be conjugated to an internal position of the sense strand.
  • the in vivo delivery enhancing moiety is not conjugated to an external position of the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to the antisense strand.
  • the in vivo delivery enhancing moiety may be conjugated to an external position of the antisense25 strand, e.g., the 3’-end of the antisense strand, to the 5’-end of the antisense strand, or to both the 5- end and the 3’-end of the antisense strand.
  • the in vivo delivery enhancing 44 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO moiety may be conjugated to an internal position of the antisense strand.
  • the in vivo delivery enhancing moiety is not conjugated to an external position of the antisense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand and the in vivo delivery enhancing moiety are both conjugated to the sense strand of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin 5 targeting ligand is conjugated to an external position of the sense strand, e.g., the 3’-end of the sense strand or the 5’-end of the sense strand
  • the in vivo delivery enhancing moiety is conjugated to an internal position of the sense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand and the in vivo delivery enhancing moiety are both conjugated to the antisense strand of the dsRNA agent.
  • the ⁇ v ⁇ 6 10 integrin targeting ligand is conjugated to an external position of the antisense strand, e.g., the 3’-end of the sense strand or the 5’-end of the antisense strand, and the in vivo delivery enhancing moiety is conjugated to an internal position of the antisense strand.
  • the ⁇ v ⁇ 6 integrin targeting ligand is conjugated to a sense strand, and the in vivo delivery enhancing moiety is conjugated to an antisense strand.
  • the 15 ⁇ v ⁇ 6 integrin targeting ligand is conjugated to an antisense strand, and the in vivo delivery enhancing moiety is conjugated to a sense strand.
  • the dsRNA agent includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a DMPK gene.
  • the region of complementarity is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).
  • the iRNA Upon contact with a cell expressing the DMPK gene, the iRNA inhibits the expression of the DMPK gene (e.g., a human, a primate, a non-primate, or a rat gene) by at least about 50% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flow cytometric techniques.
  • inhibition of expression is determined by the qPCR method 25 provided in the examples herein with the siRNA at, e.g., a 10 nM concentration, in an appropriate organism cell line provided therein.
  • inhibition of expression in vivo is determined by knockdown of the human gene in a rodent expressing the human gene, e.g., a mouse or an AAV-infected mouse expressing the human target gene, e.g., when administered as single dose, e.g., at 3 mg/kg at the nadir of RNA expression.
  • a dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used.
  • One strand of a dsRNA (the antisense s trand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence.
  • the target sequence can be derived from the sequence of an mRNA formed during the expression of a DMPK gene.
  • the other strand includes a 35 region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
  • the complementary sequences of a dsRNA can also be contained as self- 45 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
  • the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15- 26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 5 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19- 22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.
  • the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22- 10 25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 basepairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.
  • the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15- 15 17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20- 24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for example 19-23 nucleotides in length or 21-23 nucleotides in length.
  • the duplex structure is 19 to 30 base pairs in length.
  • the region of complementarity to the target sequence is 19 to 30 nucleotides in length.
  • the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length.
  • the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 25 nucleotides in length may serve as substrates for Dicer.
  • RNAi-directed cleavage i.e., cleavage through a RISC pathway
  • the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20- 25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs.
  • an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA.
  • a miRNA is a dsRNA.
  • a 46 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO dsRNA is not a naturally occurring miRNA.
  • an iRNA agent useful to target DMPK gene expression is not generated in the target cell by cleavage of a larger dsRNA.
  • a dsRNA as described herein can further include one or more single-stranded nucleotide overhangs, e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide 5 overhang can have superior inhibitory properties relative to their blunt-ended counterparts.
  • a nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5'-end, 3'- end, or both ends of an antisense or sense strand of a dsRNA. 10
  • a dsRNA can be synthesized by standard methods known in the art. Double stranded RNAi compounds of the disclosure may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both.
  • a dsRNA of the disclosure includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence.
  • the sense strand is selected from the group of sequences 20 provided in any one of Tables 2-3, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 2-3.
  • one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a-associated target gene.
  • a dsRNA will include two oligonucleotides, where one 25 oligonucleotide is described as the sense strand in any one of Tables 2-3, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-3.
  • the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides.
  • the substantially complementary 30 sequences of the dsRNA are contained on a single oligonucleotide.
  • the RNA of the iRNA of the disclosure e.g., a dsRNA of the disclosure, may comprise any one of the sequences set forth in any one of Tables 2-3 that is un- modified, un-conjugated, or modified or conjugated differently than described therein.
  • the disclosure encompasses dsRNA of Tables 2-3 which are un-modified, un-conjugated, modified, or conjugated, as described herein.
  • dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference 47 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO (Elbashir et al., EMBO 2001, 20:6877-6888).
  • ALN-529-WO Elbashir et al., EMBO 2001, 20:6877-6888.
  • others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).
  • dsRNAs described herein can include at 5 least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having any one of the sequences in any one of Tables 2-3 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above.
  • dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides derived from any one of the sequences of any one of Tables 2-3, and differing in their ability to inhibit the expression of a 10 DMPK gene by not more than about 5, 10, 15, 20, 25, or 30 % inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present disclosure.
  • the RNAs provided in Tables 2-3 identify a site(s) in a DMPK transcript that is susceptible to RISC-mediated cleavage. As such, the present disclosure further features iRNAs that target within one of these sites.
  • an iRNA is said to target within a particular site of an 15 RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site.
  • Such an iRNA will generally include at least about 19 contiguous nucleotides from any one of the sequences provided in any one of Tables 2-3 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a DMPK gene. 20 III. Modifications for the RNAi Agents of the Disclosure
  • the iRNA for use in the methods of the disclosure e.g., a dsRNA, is un- m odified, and does not comprise, e.g., chemical modifications and/or conjugations known in the art and described herein.
  • the iRNA for use in the methods of the disclosure is chemically modified to enhance stability or other beneficial characteristics.
  • substantially all of the nucleotides of an iRNA of the disclosure are modified.
  • all of the nucleotides of an iRNA of the disclosure are modified.
  • iRNAs of the disclosure in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.
  • substantially all of the nucleotides of an iRNA of the disclosure 30 are modified and the iRNA agents comprise no more than 10 nucleotides comprising 2’-fluoro modifications (e.g., no more than 92 ⁇ -fluoro modifications, no more than 82 ⁇ -fluoro modifications, no more than 72 ⁇ -fluoro modifications, no more than 62 ⁇ -fluoro modifications, no more than 52 ⁇ -fluoro modifications, no more than 42 ⁇ -fluoro modifications, no more than 52 ⁇ -fluoro modifications, no more than 42 ⁇ -fluoro modifications, no more than 32 ⁇ -fluoro modifications, or no more than 22 ⁇ -fluoro 35 modifications).
  • 2’-fluoro modifications e.g., no more than 92 ⁇ -fluoro modifications, no more than 82 ⁇ -fluoro modifications, no more than 72 ⁇ -fluoro modifications, no more than 62 ⁇ -fluoro modifications, no more than 52 ⁇ -fluoro modifications, no more than 42 ⁇ -fluoro modifications, no more than 52
  • the sense strand comprises no more than 4 nucleotides comprising 2 ⁇ -fluoro modifications (e.g., no more than 32 ⁇ -fluoro modifications, or no more than 22 ⁇ -fluoro modifications).
  • the antisense strand comprises no more than 6 nucleotides comprising 2 ⁇ -fluoro modifications (e.g., no more than 52 ⁇ -fluoro modifications, no more than 48 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 42 ⁇ -fluoro modifications, no more than 42 ⁇ -fluoro modifications, or no more than 22 ⁇ -fluoro modifications).
  • all of the nucleotides of an iRNA of the disclosure are modified and the iRNA agents comprise no more than 10 nucleotides comprising 2’-fluoro modifications (e.g., no 5 more than 92 ⁇ -fluoro modifications, no more than 82 ⁇ -fluoro modifications, no more than 72 ⁇ -fluoro modifications, no more than 62 ⁇ -fluoro modifications, no more than 52 ⁇ -fluoro modifications, no more than 42 ⁇ -fluoro modifications, no more than 52 ⁇ -fluoro modifications, no more than 42 ⁇ -fluoro modifications, no more than 32 ⁇ -fluoro modifications, or no more than 22 ⁇ -fluoro modifications).
  • 2’-fluoro modifications e.g., no 5 more than 92 ⁇ -fluoro modifications, no more than 82 ⁇ -fluoro modifications, no more than 72 ⁇ -fluoro modifications, no more than 62 ⁇ -fluoro modifications, no more than 52 ⁇ -fluoro modifications, no more than 42 ⁇ -fluoro modifications, no more than 52 ⁇
  • nucleic acids featured in the disclosure can be synthesized and/or modified by methods well 10 established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, end modifications, e.g., 5’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or 15 bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or 4’-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.
  • base modifications e.g., replacement with stabilizing bases, destabilizing bases, or 15 bases that base pair with an expanded repertoire of partners, removal of bases (abasic
  • RNAs having 20 modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • a modified iRNA will have a phosphorus atom in its internucleoside backbone.
  • Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, 25 phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' 30 to 5'-2'.
  • the dsRNA agents of the disclosure are in a free acid form. In other embodiments of the disclosure, the dsRNA agents of the disclosure are in a salt form. In one embodiment, the dsRNA agents of the disclosure are in a sodium salt form. In certain embodiments, when the dsRNA agents of the disclosure are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester and/or 35 phosphorothiotate groups present in the agent.
  • Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion include not more than 5, 4, 3, 2, or 1 phosphodiester and/or phosphorothioate linkages without a sodium counterion.
  • sodium ions are present in the agent as counterions for all of the phosphodiester and/or phosphorothiotate groups present in the agent.
  • Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S.
  • Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • patents that teach the preparation of the above oligonucleosides include, but 20 are not limited to, U.S. Patent Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.
  • RNA mimetics are contemplated for use in iRNAs, in which both 25 the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a peptide nucleic acid PNA
  • PNA peptide nucleic acid
  • the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine 30 backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos.5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the disclosure are described in, for example, in Nielsen et al., Science, 1991, 254, 35 1497-1500.
  • RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular --CH2--NH--CH2-, --CH2--N(CH3)--O-- CH2--[known as a methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2-- 50 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Patent No.5,034,506. 5 Modified RNAs can also contain one or more substituted sugar moieties.
  • the iRNAs e.g., dsRNAs, featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
  • Exemplary suitable modifications include O[(CH2)nO] mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and 10 O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10.
  • dsRNAs include one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of 15 an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties.
  • the modification includes a 2'-methoxyethoxy (2'-O-- CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • 2'-dimethylaminooxyethoxy i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below
  • 2'- 20 dimethylaminoethoxyethoxy also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE
  • 2'-O--CH2--O--CH2--N(CH2)2 2'-dimethylaminooxyethoxy
  • modifications include : 5’-Me-2’-F nucleotides, 5’- Me-2’-OMe nucleotides, 5’-Me-2’-deoxynucleotides, (both R and S isomers in these three families); 2’- alkoxyalkyl; and 2’-NMA (N-methylacetamide).
  • Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 25 2'-fluoro (2'-F).
  • RNA of an iRNA can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat.
  • nucleobase of the disclosure can also include nucleobase (often referred to in the art simply as 35 “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5- me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl 51 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • nucleobases include those disclosed in U.S. Pat. No.3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, 10 Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993.
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N- 15 2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 °C (Sanghvi, Y. S., Crooke, S. T.
  • An iRNA of the disclosure can also be modified to include one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in 30 the 3'-endo structural conformation.
  • the addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A.
  • An iRNA of the disclosure can also be modified to include one or more bicyclic sugar moities.
  • a 35 “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms.
  • A“bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′- carbon and the 2′-carbon of the sugar ring.
  • an agent of the disclosure may 52 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO include one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons.
  • an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural conformation.
  • bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms.
  • the antisense polynucleotide agents of the disclosure include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge.
  • 4′ to 2′ bridged bicyclic nucleosides include but are not limited to 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2′; 4′-(CH2)2—O-2′ (ENA); 4′-CH(CH3)—O- 2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)—O-2′ (and analogs thereof; see, e.g., U.S. Pat.
  • any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example ⁇ -L-ribofuranose and ⁇ -D-ribofuranose (see WO 99/14226).
  • An iRNA of the disclosure can also be modified to include one or more constrained ethyl 30 nucleotides.
  • a "constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge.
  • an iRNA of the disclosure comprises one or more monomers that are UNA 5 (unlocked nucleic acid) nucleotides.
  • UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked "sugar" residue.
  • UNA also encompasses monomer with bonds between C1'-C4' have been removed (i.e. the covalent carbon-oxygen- carbon bond between the C1' and C4' carbons).
  • the C2'-C3' bond i.e. the covalent carbon-carbon bond between the C2' and C3' carbons
  • the antisense strand comprises at least one (e.g., one, two, three, four, five or more) 54 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5’ region of the antisense strand.
  • one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5’- end of the antisense strand.
  • the thermally destabilizing modification(s) 5 of the duplex is/are located at position 6, 7 or 8 from the 5’-end of the antisense strand.
  • the thermally destabilizing modification of the duplex is located at position 7 from the 5’-end of the antisense strand.
  • the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5’-end of the antisense strand.
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are independently or in combination absent from the nucleotide.
  • bonds between the ribose carbons e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’
  • acyclic nucleotide 56 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO is a modified or unmodified nucleobase, R 1 and R 2 independently are H, halogen, OR 3 , or alkyl; and R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • the term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked 5 "sugar" residue.
  • UNA also encompasses monomers with bonds between C1'-C4' being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons).
  • bonds between C1'-C4' being removed i.e. the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons.
  • the C2'-C3' bond i.e. the covalent carbon-carbon bond between the C2' and C3' carbons
  • the acyclic derivative provides greater backbone flexibility without affecting the Watson- Crick pairings.
  • the acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.
  • the term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds: 15 .
  • the thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex.
  • mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof.
  • Other 20 mismatch base pairings known in the art are also amenable to the present disclosure.
  • a mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides.
  • the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy 25 nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand.
  • 57 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as: .
  • thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.
  • the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand.
  • the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more ⁇ -nucleotide complementary to the base on the target mRNA, such as: 5 wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.
  • the alkyl for the R group can be a C1-C6alkyl.
  • Specific alkyls for the R group include, but are 10 not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.
  • nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into an RNAi agent of the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the 15 range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides.
  • the dsRNA agent further comprises a phosphate or phosphate mimic at the 5’-end of the sense or antisense strand. In one embodiment, there is a phosphate or phosphate mimic at the 5’-end of the sense strand. In one embodiment, there is a phosphate or phosphate mimic at the 5’-end of the antisense strand.
  • the phosphate mimic is 5’-end phosphorothioate (5’-PS), 5’- 25 end phosphorodithioate (5’-PS2), 5’ end vinylphosphonate (5’-VP), 5’-end m ethylphosphonate (MePhos), or 5’-deoxy-5’-C-malonyl one embodiment, the phosphate mimic is a 5’-vinyl phosphonate (VP).
  • the phosphate mimic is a 5’-(E)-vinyl phosphonate (VP) isomer (i.e., trans-vinylphosphate), 5’-(Z)-VP isomer (i.e., cis-vinylphosphate), or mixtures thereof.
  • VP 5’-(E)-vinyl phosphonate
  • 5’-(Z)-VP isomer i.e., cis-vinylphosphate
  • 59 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the -CH2OH group at the 4’-position of the 5’-terminal nucleotide is 15 replaced with a phosphate mimic of the formula -O-CH2-P(O)(OR)2, wherein each R is independently hydrogen or C1-4 alkyl (e.g., one R group is hydrogen and one R group is methyl; or both R groups are hydrogen).
  • the phosphate mimic is a 5’-cyclopropyl phosphonate (VP) (i.e., the CH 2 OH group at the 4’-position of the 5’-terminal nucleotide is replaced with a group of the formula -Cy- 20 P(O)(OR) 2 , wherein Cy is a cyclopropyl ring and each R is independently hydrogen or C 1-4 alkyl (e.g., one R group is hydrogen or both R groups are hydrogen).
  • the 5’-end phosphate mimic i , salt (e.g., sodium salt) thereof, wherein B is an optionally modified nucleobase (e.g., U).
  • the 5’-end phosphate mimic is part of a modified 5’-terminal nucleotide.
  • the phosphate mimic may be part of a modified 5’-terminal nucleotide 60 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO having the structure , wherein B is an optionally modified nucleobase.
  • the 5’-end phosphate mimic can also include a 5’-phosphate prodrug or 5’-phosphonate prodrug.
  • the 5’-phosphate prodrug or 5’-phosphonate 5 prodrug has a structure of formulas disclosed in WO2022/147214, which is incorporated herein by reference.
  • the 5’-phosphate prodrug or 5’-phosphonate prodrug is: 10 -methoxyphenyl)-3,3-dimethyl-1,2-dithiolan-4-ol) 61 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • the 5’-phosphate prodrug or 5’-phosphonate prodrug is: .
  • the siRNA containing one of the above list of 5’ modified phosphate prodrugs generally has an activity comparable to that of the siRNA containing 5’-VP.
  • the 5’- 5 phosphate prodrug or 5’-phosphonate prodrug is: .
  • the siRNA containing one of the above list of 5’ modified phosphate prodrugs can have an improved stability than that of the siRNA containing 5’-VP and better or comparable activity than that of the siRNA containing 5’-VP.
  • the 5’-end of the antisense strand of the dsRNA agent does not contain a 5’-vinyl phosphonate (VP). IV.
  • a dsRNA agent comprises an antisense strand and a sense strand; at least one ⁇ v ⁇ 6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand.
  • Suitable in vivo delivery enhancing moieties for use in the present disclosure are described in 20 International Application No. PCT/US2022/046668, filed on October 14, 2022, in U.S.
  • the at least one in vivo delivery enhancing moiety may be conjugated to any internal position of the antisense strand or the sense strand. In some embodiments, the at least one in vivo delivery enhancing moiety may be conjugated to a position that is not an external position of the antisense strand or the sense strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to an external positon of the antisense strand or the sense strand.
  • the term “in vivo delivery enhancing moiety” refers to a moiety which, when conjugated to a dsRNA agent, enhances delivery of the dsRNA agent to a target tissue, e.g., muscle, 62 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO or a cell type, such as a muscle cell (e.g., a skeletal muscle cell or a cardiac muscle cell).
  • the in vivo delivery enhancing moiety has pharmacokinetic (PK) enhancing properties, such as increasing the residence time in the blood of the dsRNA agent, and/or increasing tissue PK, including increasing tissue exposure of the dsRNA agent.
  • PK pharmacokinetic
  • the in vivo delivery 5 enhancing moiety can have a cooperative or synergistic effect with an ⁇ v ⁇ 6 compound in increasing tissue exposure when both elements are conjugated to the dsRNA agent.
  • the in vivo delivery enhancing moiety is lipophilic.
  • the in vivo delivery enhancing moiety increases lipophilicity of the dsRNA agent and provides optimal 10 hydrophobicity for the enhanced in vivo delivery of dsRNA to muscle tissue, e.g., skeletal muscle tissue or cardiac muscle tissue.
  • muscle tissue e.g., skeletal muscle tissue or cardiac muscle tissue.
  • One way to characterize lipophilicity is by the octanol-water partition coefficient, logKow, where Kow is the ratio of a chemical’s concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium.
  • the octanol-water partition coefficient is a 15 laboratory-measured property of a substance.
  • a chemical substance is lipophilic in character when its logKow exceeds 0.
  • the lipophilic moiety possesses a logKow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10.
  • the logKow of 6- amino hexanol for instance, is predicted to be approximately 0.7.
  • the logKow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7. 25
  • the lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a hydrocarbon chain, e.g., a C22 hydrocarbon chain, can increase or decrease the partition coefficient (e.g., logKow) value of the hydrocarbon chain.
  • the hydrophobicity of the dsRNA agent conjugated to at least one in vivo 30 delivery enhancing moiety can be measured by its protein binding characteristics.
  • the unbound fraction in the plasma protein binding assay of the dsRNA agent can be determined to positively correlate to the relative hydrophobicity of the dsRNA agent, which can positively correlate to the silencing activity of the dsRNA agent.
  • the plasma protein binding assay determined is an electrophoretic 35 mobility shift assay (EMSA) using human serum albumin protein.
  • ESA electrophoretic 35 mobility shift assay
  • the hydrophobicity of the dsRNA agent measured by fraction of unbound dsRNA agent in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of dsRNA agent.
  • the at least one in vivo delivery enhancing moiety may be attached to the dsRNA agent of the disclosure by any method known in the art, including via a functional grouping already present in the in vivo delivery enhancing moiety or introduced into the dsRNA agent, such as a hydroxy group (e.g., —CO—CH2—OH).
  • the functional groups already present in the in vivo delivery enhancing moiety 5 or introduced into the dsRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. Conjugation of the dsRNA agent and the in vivo delivery enhancing moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy and an alkyl group R—, an alkanoyl group RCO— or a substituted carbamoyl group 10 RNHCO—.
  • the alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained or branched; and saturated or unsaturated).
  • Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like.
  • more than one in vivo delivery enhancing moiety can be conjugated 15 to the dsRNA agent of the disclosure.
  • two or more in vivo delivery enhancing moieties are conjugated to the same strand of the dsRNA agent.
  • each strand of the dsRNA agent is conjugated to one or more in vivo delivery enhancing moieties.
  • two or more in vivo delivery enhancing moieties are conjugated to the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the dsRNA agent.
  • the in vivo delivery enhancing moiety may comprise at least one C10- 25 C26 hydrocarbon chain, e.g., a C10 hydrocarbon chain, a C11 hydrocarbon chain, a C12 hydrocarbon chain, a C13 hydrocarbon chain, a C14 hydrocarbon chain, a C15 hydrocarbon chain, a C16 hydrocarbon chain, a C17 hydrocarbon chain, a C18 hydrocarbon chain, a C19 hydrocarbon chain, a C20 hydrocarbon chain, a C21 hydrocarbon chain, a C22 hydrocarbon chain, a C23 hydrocarbon chain, a C24 hydrocarbon chain, a C25 hydrocarbon chain or a C26 hydrocarbon chain.
  • a C10 hydrocarbon chain e.g., a C10 hydrocarbon chain, a C11 hydrocarbon chain, a C12 hydrocarbon chain, a C13 hydrocarbon chain, a C14 hydrocarbon chain, a C15 hydrocarbon chain, a C16 hydrocarbon chain, a C17 hydrocarbon chain, a C18 hydrocarbon chain,
  • the C10-C26 30 hydrocarbon chain may be a straight hydrocarbon chain. In other embodiments, the C10-C26 hydrocarbon chain may be a branched hydrocarbon chain. In some embodiments, the C10-C26 hydrocarbon chain may be a saturated hydrocarbon chain. In other embodiments, the C10-C26 hydrocarbon chain may be an unsaturated hydrocarbon chain, e.g., comprising one or more double bonds and/or one or more triple bonds. 35
  • the in vivo delivery enhancing moiety may comprise at least one C11 hydrocarbon chain. In one embodiment, the at least one in vivo delivery enhancing moiety may comprise at least one C17 hydrocarbon chain.
  • the in vivo delivery enhancing moiety may comprise at least one C22 hydrocarbon chain. 64 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • the C10-C26 hydrocarbon chain may be unsubstituted.
  • the C10-C26 hydrocarbon chain may be substituted with at least one functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.
  • the C10-C26 hydrocarbon chain is substituted with a carboxylic 5 acid group.
  • the C10-C26 hydrocarbon chain is represented by –(CH2)n-, wherein n is a number from 10 to 26, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26; and — is a bond connecting the C10-C26 hydrocarbon chain to other portions of the in vivo delivery enhancing moiety.
  • the C10-C26 hydrocarbon chain is connected at both ends to the 10 remainder of the in vivo delivery enhancing moiety.
  • the C10-C26 hydrocarbon chain is represented by the following structure: –linker-(CH2)n(CH3), wherein n is a number from 10 to 25, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; the linker is as described hereinbelow; and – is a bond connecting the C10-C26 hydrocarbon chain to other portions of the in vivo delivery enhancing moiety.
  • the C10-C26 hydrocarbon chain is connected at one end to other portions of the in vivo delivery enhancing moiety.
  • the C10-C26 hydrocarbon chain is represented by the following structure: –linker-(CH2)n-A, wherein n is a number from 10 to 26, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26; the linker as described hereinbelow and A is a functional group 20 selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne; or A1 or A2 with a structure shown below: In one embodiment, A is carboxylic acid. 25 In one embodiment, A is A1. In one embodiment, A is A2.
  • in vivo delivery enhancing moieties useful in the context of the present disclosure are described, e.g., in WO 2023/064530, the entire contents of which are incorporated herein by reference.
  • the in vivo delivery enhancing moiety an comprise at least one C10-C26 h ydrocarbon chain.
  • the in vivo delivery enhancing moiety comprises a C22 hydrocarbon chain, e.g., one or more C22 hydrocarbon chains.
  • the C22 hydrocarbon chain is a C22 acid, e.g., a C22 acid selected from the group consisting of docosanoic acid, 5 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-cis-7,10,13,16,19-docosapentaenoic acid, all-cis-4,7,10,13,16,19-docosahexaenoic acid, all-cis-13,16-docosadienoic acid, all-cis-7,10,13,16- docosatetraenoic acid, all-cis-4,7,10,13,16-docosapentaenoic acid, and cis-13-docosenoic acid.
  • a C22 acid selected from the group consisting of docosanoic acid, 5 6-octyltetradecanoic acid, 10-hexylhexadecanoic acid, all-
  • the C 22 hydrocarbon chain is a C 22 alcohol, e.g. the C 22 alcohol selected10 from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13- docosen-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,10,13,16,19- docosahexanol.
  • the C 22 alcohol selected10 from the group consisting of 1-docosanol, 6-octyltetradecan-1-ol, 10-hexylhexadecan-1-ol, cis-13- docosen-1-ol, docosan-9-ol, docosan-2-ol, docosan-10-ol, docosan-11-ol, and cis-4,7,
  • the C22 hydrocarbon chain is a C22 amide, e.g., the C22 amide selected 15 from the group consisting of (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Docos-11-enamide,12-Docosenamide, (Z)-Docos-13-enamide, (Z)-N-Hydroxy-13- docoseneamide, (E)-Docos-14-enamide, 6-cis-Docosenamide, 14-Docosenamide Docos-11-enamide, (4E,13E)-Docosa-4,13-dienamide, and (5E,13E)-Docosa-5,13-dienamide.
  • the C22 amide selected 15 from the group consisting of (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)-Docos-9-enamide, (E)-Doco
  • the C22 hydrocarbon chain includes, but are not limited to, docosan-2-yl, 20 docosan-3-yl, docosan-4-yl, docosan-5-yl, docosan-6-yl, docosan-7-yl, docosan-8-yl, docosan-9-yl, docosan-10-yl, docosan-11-yl, 2-(decyl)dodecan-1-yl, 2-(nonyl)tridecan-1-yl, 2-(octyl)tetradecan-1- yl, 2-(heptyl)pentadecan-1-yl, 2-(hexyl)hexadecan-1-yl, 2-(pentyl)heptadecan-1-yl, 2- 66 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the at least one C10-C26 hydrocarbon chain comprised in the in vivo delivery enhancing moiety may be conjugated to the remainder of the dsRNA agent via a linker or via a carrier or via an internucleotide phosphate linkage.
  • the at least one C10-C26 hydrocarbon chain is conjugated to the remainder of the dsRNA agent via a linker.
  • the linker may be a cleavable linker, i.e., the linker comprises a portion that can be 20 cleavable chemically or enzymatically.
  • amide linkages can be enzymatically cleavable.
  • the linker comprises —(CH2)n-NH-C(O)- or –(CH2)n-NH-C(O)-(CH2)2- C(COOH)-NH-C(O)-, wherein n is a number from 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • the linker comprises –(CH)2-O-(CH2CH2)-(O)- (CH2CH2)-NH-C(O)-.
  • the linker may comprise a group selected from the group consisting of an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, and a carbamate.
  • the linker may be selected from the group consisting of -(CH2)nNH-; -C(O)(CH2)nNH-; -NR’’’’(CH2)nNH-, -C(O)-(CH2)n-C(O)-; -C(O)-(CH2)n-C(O)O-; -C(O)-O-; -C(O)-(CH2)n-NH-C(O)-; 30 -C(O)-(CH2)n-; -C(O)-NH-; -C(O)-; -(CH2)n-C(O)-; -(CH2)n-C(O)O-; -(CH2)n-; and -(CH2)n-NH- C(O)-; wherein n is a number from 1 to 20; and R’’’’ is C1-C6 alkyl, e.g., C1 alkyl, C2 alkyl, C3 alkyl
  • n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19 or 20.
  • the ⁇ v ⁇ 6 integrin targeting ligand is linked to the remainder of the 35 dsRNA agent as shown in the exemplary schematics below, wherein “2’-N6” refers to attachment of a lipophilic moiety (e.g., C22) at the 2’ position on the nucleotide at position 6 on the sense strand (counting from the 5’ end): 67 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the in vivo delivery enhancing moiety that may be conjugated to the 15 dsRNA agent of the disclosure is represented by the following structure: , 20 , 69 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5 wherein the broken bond represents the bond to the remainder of the dsRNA agent (e.g., to a 2’- 10 oxygen of a nucleoside).
  • the in vivo delivery enhancing moiety may be attached to the remainder of the dsRNA agent at a 2’ position on an internal nucleotide.
  • the at least one in vivo delivery enhancing moiety may be conjugated to the dsRNA agent via a direct attachment to the ribosugar of the dsRNA agent, e.g., an antisense strand or a sense strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to the dsRNA agent via a linker or a carrier.
  • at least one in vivo delivery enhancing moiety may be conjugated to the iRNA agent via one or more linkers (tethers).
  • the at least one in vivo delivery enhancing moiety is conjugated to the20 dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide- thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.
  • a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide- thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.
  • the in vivo delivery enhancing moiety and the targeting moiety are independently present within: (a) an internally-modified nucleosides such as, 5 (b) a modified internucleotide linkage such as, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), (c) a 5’-terminal modification such as 10 C(O), S(O) 2 , or -P(Y’)(OH)-O-; or (ii) -P(Y)(OH)O-R L or -C(O)N(H)R L , wherein Y is O or S; (d) a 3’-terminal modification such as -P(Y)(OH)-R 3 , wherein Y is O or S; and R 3 is 15 wherein: B is an optionally modified nucleobase; 71 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Aln
  • ALN-529-WO B 1 is a nucleobase modified with a lipophilic moiety or a targeting moiety (e.g., a pyrimidine nucleobase modified at the 5-position);
  • R L , R L1 , and R L2 are each a group containing a lipophilic moiety or a targeting moiety;
  • R 2 ’ or R 3 ’ may be any functional group that is an acceptable 2’-modification for a ribose 5 sugar.
  • R 2 ’ or R 3 ’groups include, but are not limited to, hydrogen, halogen (e.g., 2’-fluoro), hydroxy, 2’-O-alkyl (e.g., 2’-OMethyl), 2’-O-methoxyalkyl (e.g., 2’-O-methoxymethyl, 2’-O-methoxyethyl, or 2’-O-2-methoxypropanyl) modification, 2’-O-allyl modification, 2’-C-allyl modification, 2'-O-N-methylacetamido (2'-O-NMA, i.e.
  • halogen e.g., 2’-fluoro
  • hydroxy hydroxy
  • 2’-O-alkyl e.g., 2’-OMethyl
  • 2’-O-methoxyalkyl e.g., 2’-O-methoxymethyl, 2’-O-methoxyethyl, or 2
  • R 2 ’ or R 3 ’ may be H, OH, F, OMe, O-methoxyalkyl, O-allyl, O-N-methylacetamido, O-dimethylaminoethoxyethyl, or O-aminopropyl.
  • R L , R L1 , and R L2 are each a group containing a lipophilic moiety, such as a saturated or unsaturated C22-hydrocarbon chain.
  • R L when R L comprises a lipophilic moiety, then R L can be selected from the group consisting of: 15 wherein integer m is 0-8 (e.g., 0; or 1-8; or 0-6; or 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (e.g., 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); W is C 1 -C 4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or t-butyl); R and R’ are each independently H or C 1 -C 4 alkyl (e.g., methyl, e
  • ALN-529-WO the group consisting of halogen, -OR G , -SR G , -N(R G ) 2 , -C(O)OR G , -OC(O)R G , -C(O)N(R G ) 2 , - N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO 2 (R G ), or -SO 2 N(R G ) 2 , wherein each R G is independently hydrogen or C 1- C 6 alkyl (for instance, G is optionally substituted with a -OR G , -C(O)OR G , or - N(R G )C(O)R G ); and 5 G 1 is a saturated or unsaturated C 22 hydrocarbon chain (for instance, G 1 may be a linear or branched C 22 alkyl group), wherein G 1 is optionally substituted with one or two groups selected from the group consist
  • E xamples of RL include, but are not limited to the following structures: F urther examples of RL include, but are not limited to the following structures: , 73 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO ,
  • R L1 can be selected from the group consisting of
  • R L2 can be selected from the group consisting of the following 5 structures: 74 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • integer m is 0-8 (for instance, m is 0; or m is 1-8; or m is 0-6; or m is 1; or 2; or 3; or 4; or 5; or 6; or 7; or 8); integer n is 1-21 (for instance, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2; or 2 or 3 or 4 or 5 or 6); 5 R and R’ are each independently H or an alkyl group such as a C1-C4 alkyl (e.g., methyl, ethyl, propyl, isopropyl, t-butyl); G is G1 or a saturated or unsaturated C21 hydrocarbon chain (i.e., G together with the carbonyl to which it is attached may form a group with 22 carbons) (for instance, G may be a linear or branched C 21 alkyl group), wherein G is optionally substituted with one or two groups selected from10 the group consisting of hal
  • B 1 is a nucleobase modified with a G or G 1 group, wherein G and G 1 are as defined above (e.g., a pyrimidine nucleobase modified at the 5’-position with a group c omprising G or G1).
  • B1 include, but are not limited to, .
  • in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: wherein: B is an optionally modified nucleobase; 10 G 3 is a saturated or unsaturated C 1-20 hydrocarbon group (e.g., C 1-6 alkylene; C 2-6 alkylene; or hexylene); L K is a linking group such as -O-, -N(H)-, -S-, -S-S-, -C(O)O-, OC(O)-, -C(O)N(H)-, - N(H)C(O), -OC(O)N(H)-, -N(H)C(O)O-, -S(O) 2 -, -S(O) 2 O-, -S(O) 2 N(H)-, -P(O)(OH)O-, - OP(O)(OH)-, -P(S)(OH)O-, - OP(O)
  • R G is hydrogen.
  • R G is OH, 76 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • R G is COOH.
  • R G is CONH 2 .
  • R G is amino.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, 5 individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
  • a modified internucleotide linkage is shown with substituent atoms fully described at the phosphorous atom, e.g., , where C’ is the 2’-carbon or 3’-carbon atom of a ribose ring, it is understood that the oxygen having the broken bond is the 5'-oxygen of the subsequent nucleotide.
  • the in vivo delivery enhancing moiety is present within a modified n ucleoside of the formula: . In one embodiment, the in vivo delivery enhancing moiety is present within a modified n ucleoside of the formula: . In one embodiment, the in vivo delivery enhancing moiety is present within a modified 15 nucleoside of the formula: . In one embodiment, the in vivo delivery enhancing moiety is present within a modified n ucleoside of the formula: .
  • the in vivo delivery enhancing moiety is present within a modified nucleoside of the formula: 20 1-10, 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6);
  • G is a C22 hydrocarbon chain, optionally substituted with one or two groups selected from the group consisting of halogen, -OR G , -SR G , - N(R G )2, -C(O)OR G , -OC(O)R G , -C(O)N(R G )2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or - 77 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each R G is independently hydrogen or C 1- C 6 alkyl; and nucleobase B is a modified or unmodified nucleobase.
  • n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is C 22 alkyl chain.
  • the in vivo delivery enhancing moiety is present within a modified 5 nucleoside of the formula: , wherein n is an integer of 1-21, for instance, 1-12, 1- 10, 1-8, 1-6, 1-4, or 1-2, or 2 or 3 or 4 or 5 or 6); G is a C22 hydrocarbon chain, optionally substituted with one or two groups selected from the group consisting of halogen, -OR G , -SR G , -N(R G )2, - C(O)OR G , -OC(O)R G , -C(O)N(R G ) 2, -N(R G )C(O)R G , -N(R G )C(O)OR G , -N(R G )SO2(R G ), or - SO2N(R G )2, wherein each R G is independently hydrogen or C1-C6 alkyl; and nucleobase B is a 10 modified or unmodified nucleobase
  • n is 1. In one embodiment, n is 2-6. In one embodiment, n is 6. In one embodiment, G is C22 alkyl chain.
  • the in vivo delivery enhancing moiety is present within a modified n ucleoside of the formula: . In one embodiment, the in vivo delivery enhancing moiety is present within a modified 15 nucleoside of the formula: . In one embodiment, the in vivo delivery enhancing moiety is present within a modified n ucleoside of the formula: . In one embodiment, the in vivo delivery enhancing moiety is present within a modified .
  • the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(Y)(X)O-, wherein Y is O or S (e.g., O), and X is -N(H)(R L1 ), wherein R L1 is -G 1 or S(O) 2 -G 1 , each as defined above, wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • 78 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the in vivo delivery enhancing moiety is present within a modified internucleotide linkage of the form, -OP(O)(X)O-, wherein X is -N(H)(R L1 ), wherein R L1 is , wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the in vivo delivery enhancing moiety is present within a a modified internucleotide linkage of the form, wherein the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the preceding nucleotide contains a 2’-fluoro modification.
  • the preceding nucleotide contains a 2’-O-methyl modification.
  • the preceding nucleotide contains a 2’-H modification.
  • the in vivo delivery enhancing moiety is present within a a modified internucleotide linkage of the form, wherein the 3’-O is from the preceding nucleoside and the 5’-O is from the subsequent nucleoside, and wherein the phosphorous 15 atom in the internucleotide linkage is optionally enriched in the Sp or Rp isomer, or is racemic.
  • the preceding nucleotide contains a 2’-fluoro modification.
  • the preceding nucleotide contains a 2’-O-methyl modification.
  • the preceding nucleotide contains a 2’-H modification.
  • the in vivo delivery enhancing moiety is present within a 20 a modified internucleotide linkage of the form, -OP(Y)(X)O-, whereinY is O or S and X is , wherein G 1 is defined above, such .
  • in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’- end of one of the sense and antisense strands via a direct bond or through a carrier or linker.
  • in vivo delivery enhancing moiety is conjugated to the 3’-end of the sense or antisense 25 strand via a direct bond or through a carrier or linker.
  • in vivo delivery enhancing moiety is conjugated to the 5’-end of the sense or antisense strand via a direct bond or through a carrier or linker.
  • in vivo delivery enhancing moiety is conjugated to the 5’-end of one of t he sense and antisense strands and is of the formula: , or a salt thereof, wherein X is O or S (e.g., S); L is a divalent linking group (e.g., C1-20 alkyl or C1-10 alkyl-S-S-C1-10 alkyl).
  • in vivo delivery enhancing moiety is conjugated to the 5’-end of one of the 5 sense and antisense strands and is of the formula , or a salt thereof, wherein X is O or S (e.g., S).
  • R ligand is selected from the groups listed in Table R-1. Table R-1. An exemplary list of groups for R ligand 80 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula 5 . In one embodiment, in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: .
  • in vivo delivery enhancing moiety is bonded to the 3’-oxygen of the 3’- terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: 81 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • in vivo delivery enhancing moiety is conjugated to the 3’-end or 5’- end of one of the sense and antisense strands via a carrier or linker, and the carrier or linker is an inverted abasic nucleotide, such as an inverted abasic deoxyribonucleotide or an inverted abasic ribonucleotide, each connected to the remainder of the oligonucleotide via a phosphodiester (PO) or 5 phosphorothioate (PS) linkage.
  • PO phosphodiester
  • PS 5 phosphorothioate
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide and is of the formula salt thereof, wherein each10 X is independently O or S (e.g., each is S); R ligand is selected from the groups listed in Table R-1; and L is a divalent linking group (e.g., C 1-20 alkyl or C 1-10 alkyl-S-S-C 1-10 alkyl).
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula salt thereof, wherein each X is independently O or S 15 (e.g., each is S) and R ligand is selected from the groups listed in Table R-1. In some embodiments, R ligand is selected from the groups listed in Table R-2.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula 82 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO salt thereof wherein each X is independently O or S (e.g., e ach is S) and Rligand is selected from the groups listed in Table R-1, and L is a divalent linking group (e.g., C 1-20 alkyl or C 1-10 alkyl-S-S-C 1-10 alkyl.
  • in vivo delivery enhancing moiety is bonded to the 5’-oxygen of the 5’- 5 terminal nucleotide, and is of the formula salt thereof, wherein each X is O or S (e.g., each is S) and R ligand is selected from the groups listed in Table R-1.
  • R ligand is selected from the groups listed in Table R-2.
  • R L , R L1 , and R L2 are each a group containing at least one targeting 10 moiety, such as an ⁇ v ⁇ 6 integrin targeting ligand described herein.
  • the targeting moiety can be selected from:
  • the broken bond is connected to a group of the formula, **-L-ZZ-L’- , wherein ** represents the bond to the targeting ligand.
  • click adduct include those adducts formed by a copper(I)- catalyzed azide-alkyne cycloaddition reaction, a strain-promoted azide-alkyne cycloaddition, a strain- 15 promoted azide-trans-cycloalkene cycloaddition, and a thiol-maleimide Michael-addition reaction 84 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO including, for example, (i) an azide with a terminal alkyne or cycloalkyne (e.g.
  • cyclooctyne BCN, or DBCO
  • a tetrazine with a terminal alkyne or cycloalkyne e.g. cyclooctyne
  • a thiol and maleimide with or without hydrolysis of the product.
  • L and L’ are independently one of: 5 (a) -L1-[G-L2]q-G-L3-* wherein q is 0 or an integer selected from 1-10; (b) -L1-G-L2-G-L3-*; (c) -L1-G-L3-*; (d) -G-L3-*; (e) -L1-G-*; or 10 (f) -G-*.
  • L 1 is selected from one of the following groups: (a) a bond, C(O), C(S), C(NR N ), S(O)2, P(O)(OH), or P(S)(OH), wherein and R N is hydrogen or C1-6alkyl; 15 (b) a bond, C(O), P(O)(OH), or P(S)(OH); (c) a bond; (d) C(O); (e) P(O)(OH); or (f) P(S)(OH); 20 each L 2 and L 3 is independently selected from one of the following groups: ( a) -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(RN)C(O)-, -OC(O)O-, -OC(O)N(RN)-, - N(R N )C(O)O-, -N(R N )
  • each R group is independently selected from the group consisting of R’, C 1-6 alkyl, C 1- 6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, heterocyclyl, aryl, heteroaryl, C 3- 5 8 cycloalkylC 1-6 alkyl, heterocyclylC 1-6 alkyl, aryl C 1-6 alkyl, and heteroarylC 1-6 alkyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(R b ) 2 , -O(R a ), -S(R 0 ), -C(O)OR 0 ,
  • q is 0, 1, 2, 3, 4, or 5. In another embodiment of L or L’, q is 0, 1, 2, 3, or 4. In another embodiment of L or L’, q is 0, 1, 2, or 3. In another embodiment of L or L’, q is 0, 1, or 2. In another embodiment of L or L’, q is 1, 2, 3, 4, or 5, In another embodiment of L or L’, q is 1, 2, 3, or 4. In another embodiment of L or L’, q is 1, 2, or 3. In another embodiment of L or L’, q is 1 or 2. In another embodiment of L or L’, q is 4. In another embodiment of L or L’, q is 3. In 20 another embodiment of L or L’, q is 2.
  • L is one of: ( a) , wherein k is an integer from 1 to 10; L1 is bond, C(O), C(S), C(NRN), S(O)2, P(O)(OH), or P(S)(OH) (e.g., L 1 is a bond, C(O), P(O)(OH), or P(S)(OH)); and R N is hydrogen or C 1-6 alkyl; 25 (b) , wherein k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7; ( c) , wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3); 30 (d) , wherein t is an integer from 0 to 10 (e.g., an integer from 1 to 10 (e.
  • ALN-529-WO 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an integer from 3 to 7; or an integer from 4 to 6); ( e) wherein and s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, 5 an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6); independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an 10 integer from 3 to 7 or an integer from 4 to 6); or and wherein in each of the preceding embodiments of L, * represents the bond to ZZ.
  • * represents the bond to ZZ.
  • L’ is one of: (a)*-G-L 1 -, wherein L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; 15 or L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1-10alkyl; (b)*-G-[L 2 -G]q-L 1 -, wherein L 1 is a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); each L 2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein R
  • each G is independently C 1-10 alkyl or C 2-10 alkenyl, each of which is optionally substituted with 1 or 2 R groups; (f) -[G-L 2 ] q -G-*, wherein each L 2 is independently C(O)(NR N ) , N(R N )C(O), OP(O)(OH)O, or OP(S)(OH)O wherein each R N is independently hydrogen or C 1-6 alkyl; and each G is 5 independently C 1-10 alkyl, each of which is optionally substituted with 1 or 2 R groups; (g) -C 2-30 alkyl-*, such as -C 5-20 alkyl-* or -C 10-20 alkyl-*; (h) -C 1-10 alkyl-*, optionally substituted with 1 or 2 R groups; (i) -C(O)-C 2-30 alkyl-*, such as -C(O)-C
  • R L and R L1 that comprise a targeting ligand include, but are not limited to the following structures: 15 Additional examples of R L and R L1 that comprise a targeting ligand include, but are not l imited to the following structures: 88 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO Examples of R L2 that comprise a targeting ligand include, but are not limited to the following structures: A dditional examples of RL2 that comprise a targeting ligand include, but are not limited to the 5 following structures: 89 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: .
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: 90 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO .
  • R L , R L1 , and R L2 can comprise a branched linking group ( ⁇ ) capable 5 of supporting multiple targeting ligands (e.g., at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3).
  • the mutiple targeting moieties can be connected through an R L , R L1 , and R L2 of the form, (R X -L-ZZ-)z- ⁇ -T- , wherein each R X is a targeting moiety; z is at least 2; or 2-8; or 2-6; or 2-4; or 2; or 3, 10 T is -L’-T’-**, wherein ** is the bond to ⁇ , and T’ is O, S, N(H), C(O), S(O)2, C(O)N(H), N(H)C(O), OC(O), OC(O), -P(O)(OH)-, -P(S)(OH)-, -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, - P(S)(OH)O-, -OP(O)(OH)O-, or -OP(S)(OH)
  • T is selected from the following, wherein ** is the bond to ⁇ : ( a) -C(O)-X1-L5-X2-C(O)-**, wherein X1 and X2 are each independently C1-10alkyl; or C 2-10 alkyl; or C 4-10 alkyl; or C 6-10 alkyl; or C 2-8 alkyl; or C 2-6 alkyl; or C 2-4 alkyl; ( b) -C(O)-C2-20alkyl-C(O)-**, such as -C(O)-C2-12alkyl-C(O)-**, (c) -C(O)-C6-20alkyl-C(O)-**, such as -C(O)-C6-12alkyl-C(O)-**, 20 (d) -C(O)-C10alkyl-C(O)-** and (e) -C(O)-CH2CH2-C(O)-**
  • T is selected from the following, ** is the bond to ⁇ : ( f) -N(H)C(O)-C2-20alkyl-C(O)-**, (g) - N(H)C(O)-C6-20alkyl-C(O)-**, such as - N(H)C(O)-C6-12alkyl-C(O)-**, 30 (h) - N(H)C(O)-C10alkyl-C(O)-**, (i) -C(O)-C2-20alkyl-C(O)N(H)-**, 91 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • T is selected from the following, ** is the bond to ⁇ : ( a) -N(H)C(O)-X3-ZZ-X4-C(O)-**, 10 (b) -C(O)-X3-ZZ-X4-C(O)N(H)-**, (c) N(H)C(O)-X3-ZZ- X4-C(O)N(H)-**, wherein X 3 and X4 are independently C2-12alkyl; or C4-10alkyl; or C6-10alkyl; or C4-8alkyl; and ZZ is as described above (such as -C(O)N(H)-, N(H)C(O)-, -OP(O)(OH)O-, -OP(S)(OH)O-, or a click adduct).
  • T is selected from the following 15 (w) -L 6 -[G 5 -O]q5-G 5 -L 4 -**, wherein L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl; each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G 5 is independently C1-10alkyl; (x) -C(O)-[CH2CH2-O]q5-G 5 -L 4 -**, L 4 is -A 1 -B 1 -A 1 -, wherein each A 1 is independently a 20 bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1-6
  • L 4 is -A 1 -B 1 or -B 1 -A 1 -, wherein each A 1 is independently -O- or -N(H)-, and each B 1 is independently C(O), G 5 is C1-10alkyl (e.g., C2- 10alkyl or C2-6alkyl); and 25 (z) -C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-**; wherein in each of the preceding ** is the bond to ⁇ , and q5, when present, is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4.)
  • Examples of branched linking group ( ⁇ ) include, but are not limited to, 92 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-240
  • ALN-529-WO wherein the broken bond is the bond to L’.
  • Examples of -T 1 - ⁇ - include, but are not limited to, , 5
  • Examples of R L and R L1 that comprise a branched linker to a targeting ligand include, but are not limited to the following structures: 93 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • E xamples of branched RL and RL1 include, but are not limited to, the following structures: 5 94 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • branched R L2 examples include, but are not limited to, the following structures: 5 .
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: , wherein , r is an integer 10 selected from 1 - 10 (e.g., r is 7); and each R is: .
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: 95 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO .
  • targeting moiety is bonded to the 3’-oxygen of the 3’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 3 , wherein Y is O or S and R 3 is: .
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: selected from 1 - 10 (e.g., r is 7); and each R is: , wherein R X is a targeting ligand. 10
  • targeting moiety is bonded to the 5’-oxygen of the 5’-terminal nucleotide, and is of the formula -P(Y)(OH)-R 5 , wherein Y is O or S and R 5 is: ligand.
  • each R X is an integrin-receptor targeting ligand such as, .
  • the dsRNA agent comprises a double-stranded region formed between the sense and antisense strands and optionally one or two single-stranded non-loop overhang, and 5 wherein the one or more lipophilic moieties are conjugated to either the double-stranded region or the non-loop overhang.
  • the dsRNA agent does not contain a loop (e.g., stem loop) region.
  • the dsRNA agent contains a loop (e.g., stem loop) region, and the one or more lipophilic moieties are not conjugated to the loop (e.g., stem loop) region.
  • the dsRNA agent comprises a sense strand of 10 to 53 nucleotides in 10 length, in which the sense strand forms a duplex region with the antisense strand.
  • the sense strand may be 10 to 49, 12 to 49, 12 to 45, 12 to 42, 12 to 40, 15 to 49, 15 to 45, 15 to 42, 15 to 40, 15 to 38, or 15 to 36 nucleotides in length.
  • the duplex region is 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. In some embodiments, the region of complementarity to the target sequence is at least 19 contiguous 15 nucleotides in length.
  • the sense strand comprises 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 loop between S1 and S2.
  • the first 17 to 25 nucleotides counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 11 to 28 counting from 5’ end of the 20 sense strand forms a 3′-end a stem-loop set forth as: S1-L-S2.
  • the length of the stem loop S1-L-S2 is 11 to 28, 13 to 26, or 15 to 24 nucleotides in length.
  • the stem loop S1-L-S2 is 16 nucleotides in length.
  • the stem loop S1-L-S2 comprises a sequence of 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 47).
  • L is at least 3, 4, or 5 nucleotides in length. In some embodiments, L comprises a sequence of GAAA. In some embodiments, the sense strand is 36 nucleotides in length, the first 20 nucleotide counting from 5’ end of the sense strand forms a duplex region with the antisense strand, and the last 16 nucleotides forms a stem loop S1-L-S2. In one embodiment, the 16-nucleotide stem loop S1-L-S2 30 has the sequence of 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 161), wherein L is GAAA.
  • the one or more lipophilic moieties are conjugated to a non-terminal position of the sense strand. In some embodiments, the one or more lipophilic moieties are conjugated to one or more nucleotides of the stem loop S 1 -L-S 2 . In some embodiments, the one or more lipophilic moieties are 5 conjugated to one or more nucleotides of the loop L. In some embodiments, S 1 and S 2 are complementary and contain 4-10 nucleotides, e.g., S 1 and S 2 each contain 6 complementary nucleotides.
  • S 1 and S 2 are complementary and contain 4-10 nucleotides and L is GAAA, e.g., S 1 and S 2 each contain 6 complementary nucleotides and L is GAAA.
  • the one or more lipophilic moieties containing one or more saturated or unsaturated C 22 hydrocarbon chains are conjugated to one or more internal positions on at least one strand of the dsRNA agent. Dual Conjugation 15
  • in vivo delivery enhancing moiety is connected in series with a t argeting moiety, e.g., ⁇ v ⁇ 6 integrin targeting ligand, as described herein.
  • a sense or antisense strand can contain a series modification at the 3’-end or 5’-end of the oligonucleotide, 20 . such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises t he targeting moiety, e.g., ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • the series modification is of the form, wherein Q is s elected from wherein RL2 is according to any preceding 25 embodiment, wherein one of the broken bonds connects to a 5’-oxygen of a nucleoside or a 3’-oxygen of a nucleoside and the other connects to a 5’-terminal or 3’-terminal modification as described herein.
  • 98 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • a sense or antisense strand can contain a series modification of the form, wherein RL2 is according to any preceding embodiment, each Y is independently O or S; one of the broken bonds connects to a 5’-oxygen of a nucleoside or a 3’-oxygen of a nucleoside and the other connects any of 5 the 5’-terminal modifications described above or 3’-terminal modifications described above.
  • a sense or antisense strand can contain a series modification of the form, the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; each Y is independently O or S; 10 one of R 51 and R 52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety) and the other comprises a second ligand moiety (e.g., a targeting moiety, such as a ⁇ v ⁇ 6 integrin targeting ligand as described herein).
  • a lipophilic group e.g., an in vivo delivery enhancing moiety
  • a second ligand moiety e.g., a targeting moiety, such as a ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • a sense or antisense strand can contain a series modification of the 15 the broken bond connects to the 5’-oxygen of a 5’-terminal nucleoside; each Y is independently O or S; one of R 51 and R 52 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of R L or R L2 above) and the other comprises a second ligand moiety (e.g., a targeting moiety).
  • an oligonucleotide may have a series modification at the 5’-end of the formula: 99 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • a sense or antisense strand can contain a series modification of the 5 form, wherein the broken bond connects to the 3’-oxygen of a 3’-terminal nucleoside; each Y is independently O or S; one of R 31 and R 32 comprises a lipophilic group (e.g., an in vivo delivery enhancing moiety, such as any of R L2 above) and the other comprises a second ligand moiety (e.g., a targeting ligand).
  • a lipophilic group e.g., an in vivo delivery enhancing moiety, such as any of R L2 above
  • a second ligand moiety e.g., a targeting ligand
  • an oligonucleotide may have a series modification at the 3’-end of the formula: 100 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO , wherein each Y is independently O or S. 5
  • a sense or antisense strand can contain two different ligand modifications, one at the 3'-end of the strand and the other at the 5'-end of the strand: such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises t he targeting moiety, such as a ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • a sense or antisense strand can contain two different ligand modifications, one is an internal modified nuceloside or modified internucleotide linkage of the strand and the other at the 3’-end or 5'-end of the strand: 15 such that one of L1 and L2 comprises the in vivo delivery enhancing moiety and the other comprises t he targeting moiety, such as a ⁇ v ⁇ 6 integrin targeting ligand as described herein.
  • the modified 101 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO nucleoside represented by L1 can be located at a position in the strand selected from positions 2 to n- 1 , where the strand contains n nucleotides (e.g., n-1 is 20 when n is 21). For example, when L1 is at position 6, then segment (1) of the strand contains 5 nucleotides and segment (2) contains the remainder of the nucleotides within the strand.
  • L1 comprises the in vivo delivery enhancing moiety and a sense or antisense strand can be represented by one of: 10 wherein B is an optionally modified nucleobase (e.g., A, C, G, U, or T); R L or R L1 comprises the in vivo delivery enhancing moiety, L2 comprises the targeting moiety.
  • B is an optionally modified nucleobase (e.g., A, C, G, U, or T)
  • R L or R L1 comprises the in vivo delivery enhancing moiety
  • L2 comprises the targeting moiety.
  • R L is selected from the group consisting of: In one embodiment, R L1 is selected from the group consisting of: and 15 . 1 .
  • Linkers/Tethers are connected to the at least one in vivo delivery enhancing moiety at a “tethering attachment point (TAP).”
  • TAP tethering attachment point
  • Linkers/Tethers may include any C1-C100 carbon-containing 20 moiety, (e.g. C1-C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10), and may have at 102 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) group on the linker/tether, which may serve as a connection point for the lipophilic moiety.
  • Non-limited examples of linkers/tethers include TAP-(CH2)nNH-; TAP- C(O)(CH2)nNH-; TAP-NR’’’’(CH2)nNH-, TAP-C(O)-(CH2)n-C(O)-; TAP-C(O)-(CH2)n-C(O)O-; TAP- 5 C(O)-O-; TAP-C(O)-(CH2)n-NH-C(O)-; T TAP-C(O)-NH-; TAP-C(O)-; TAP- (CH2)n-C(O)-; TAP-(CH2)n-C(O)O-; TAP- CH2)n-NH-C(O)-; in which n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R’’’’ is C1-C6 alkyl.
  • n is 1-20 (e.g., 1, 2,
  • n is 5, 6, or 11.
  • the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or hydrazino group, -NHNH2.
  • the linker/tether may optionally be substituted, 10 e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S.
  • Preferred tethered ligands may include, e.g., TAP- (CH2)nNH(LIGAND); TAP-C(O)(CH2)nNH(LIGAND); TAP-NR’’’’(CH2)nNH(LIGAND); TAP- (CH2)nONH(LIGAND); TAP-C(O)(CH2)nONH(LIGAND); TAP-NR’’’’(CH2)nONH(LIGAND); TAP-(CH2)nNHNH2(LIGAND), TAP-C(O)(CH2)nNHNH2(LIGAND); TAP- 15 NR’’’(CH2)nNHNH2(LIGAND); TAP-C(O)-(CH2)n-C(O)(LIGAND); TAP-C(O)-(CH2)n- C(O)O(LIGAND); TAP-C(O)-O(LIGAND); TAP-C(O)-(CH2)n-NH-C(O)(L
  • amino terminated linkers/tethers e.g., NH2, ONH2, NH2NH2
  • the 25 tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S.
  • the double bond can be cis or trans or E or Z.
  • the linker/tether may include an electrophilic moiety, preferably at the terminal position of the linker/tether.
  • Exemplary electrophilic moieties include, e.g., an aldehyde, 30 alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester.
  • Preferred linkers/tethers include TAP- (CH2)nCHO; TAP-C(O)(CH2)nCHO; or TAP-NR’’’’(CH2)nCHO, in which n is 1-6 and R’’’’ is C1-C6 alkyl; or TAP-(CH2)nC(O)ONHS; TAP-C(O)(CH2) nC(O)ONHS; or TAP-NR’’’(CH2) nC(O)ONHS, in which n is 1-6 and R’’’’’ is C1-C6 alkyl; TAP-(CH2)nC(O)OC6F5; TAP-C(O)(CH2) nC(O) OC6F5; or 35 TAP-NR’’’’(CH2) nC(O) OC6F5, in which n is 1-11 and R’’’’ is C1-C6 alkyl; or -(CH2)nCH2LG; TAP- C(O)
  • Alnylam Reference No. ALN-529-WO Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.
  • a nucleophilic group of a ligand e.g., a thiol or amino group
  • an electrophilic group on the tether it can be desirable for the monomer to include a phthalimido group (K) a t the terminal position of the l .
  • linker/tether e.g., alloc, monomethoxy trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can be ortho-nitrophenyl or ortho, para-dinitrophenyl).
  • At least one of the linkers/tethers can be a redox cleavable linker, an acid cleavable linker, an esterase cleavable linker, a phosphatase cleavable linker, or a peptidase cleavable linker.
  • at least one of the linkers/tethers can be a reductively cleavable linker (e.g., a disulfide group).
  • At least one of the linkers/tethers can be an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group). In one embodiment, at least one of the linkers/tethers can be an esterase cleavable linker (e.g., 20 an ester group). In one embodiment, at least one of the linkers/tethers can be a phosphatase cleavable linker (e.g., a phosphate group). In one embodiment, at least one of the linkers/tethers can be a peptidase cleavable linker (e.g., a peptide bond).
  • Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood.
  • degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in 30 cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
  • a cleavable linkage group such as a disulfide bond, can be susceptible to pH.
  • the pH of 35 human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3.
  • 104 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0.
  • Some tethers will have a linkage group that is cleaved at a preferred pH, thereby releasing the iRNA agent from a ligand (e.g., a targeting or cell-permeable ligand, such as cholesterol) inside the cell, or into the desired compartment of the cell.
  • a ligand e.g., a targeting or cell-permeable ligand, such as cholesterol
  • a chemical junction that links a ligand to an iRNA agent can include a disulfide bond.
  • a disulfide bond When the iRNA agent/ligand complex is taken up into the cell by endocytosis, the acidic environment of the endosome will cause the disulfide bond to be cleaved, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res.19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol.6:466-471, 2002).
  • the ligand can be a targeting ligand or a second therapeutic agent that 10 may complement the therapeutic effects of the iRNA agent.
  • a tether can include a linking group that is cleavable by a particular enzyme.
  • the type of linking group incorporated into a tether can depend on the cell to be targeted by the iRNA agent. Tethers that contain peptide bonds can be conjugated to iRNA agents target to cell types rich in peptidases. 15
  • the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue, e.g., tissue the iRNA agent would be exposed to when administered to a subject.
  • the evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals.
  • useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
  • Redox Cleavable Linking Groups 30 One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—).
  • a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular in vivo delivery enhancing moiety, one can look to methods described herein. 35
  • a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell.
  • DTT dithiothreitol
  • the candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions.
  • candidate compounds are cleaved by at 105 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO most 10% in the blood.
  • useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).
  • the rate of cleavage of candidate compounds can be determined using standard enzyme 5 kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media. 4 .
  • Phosphate-Based Cleavable Linking Groups are cleaved by agents that degrade or hydrolyze the 10 phosphate group.
  • agents that degrade or hydrolyze the 10 phosphate group are enzymes such as phosphatases in cells.
  • phosphate-based linking groups are —O—P(O)(ORk)-O—, — O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S— P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O— P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(
  • Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, — S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S— P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(H)—S—.
  • Acid cleavable linking groups are linking groups that are cleaved under acidic conditions.
  • acid cleavable linking groups are cleaved in an acidic environment with a pH 25 of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid.
  • specific low pH organelles such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups.
  • acid cleavable linking groups include but are not limited to hydrazones, ketals, acetals, esters, and esters of amino acids.
  • Acid cleavable groups can have the general formula —C ⁇ NN—, C(O)O, or —OC(O).
  • a preferred 30 embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above. 6 .
  • Ester-Based Linking Groups 35 Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells.
  • ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups.
  • Ester cleavable linking groups have the general formula — 106 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above. 7 .
  • Peptide-Based Cleaving Groups 5 Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells.
  • Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—).
  • the amide group can be formed between any alkylene, alkenylene or alkynelene.
  • a peptide bond is a special type of amide bond formed between amino 10 acids to yield peptides and proteins.
  • the peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group.
  • Peptide cleavable linking groups have the general formula —NHCHR 1 C(O)NHCHR 2 C(O)—, where R 1 and R 2 are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. 15 8.
  • Biocleavable linkers/tethers The linkers can also include biocleavable linkers that are nucleotide and non-nucleotide linkers or combinations thereof that connect two parts of a molecule.
  • the non-nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and 20 derivatives thereof, aliphatic, alicyclic, hetercyclic, and combinations thereof.
  • At least one of the linkers is a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.
  • the bio-cleavable carbohydrate linker may have 1 to 10 saccharide units, which have at least one anomeric linkage capable of connecting two siRNA units. When two or more saccharides are present, these units can be linked via 1-3, 1-4, or 1-6 sugar linkages, or via alkyl chains.
  • Exemplary bio-cleavable linkers include: 107 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5 108 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 109 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO . 5 More discussion about the biocleavable linkers may be found in WO2018136620, entitled “Endosomal Cleavable Linkers,” the entire contents of which are incorporated herein by reference. 9.
  • the at least one in vivo delivery enhancing moiety is conjugated to 10 the dsRNA agent via a carrier that replaces one or more nucleotide(s).
  • the carrier can be a cyclic group or an acyclic group.
  • the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin.
  • the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.
  • the carrier replaces one or more nucleotide(s) in the internal position(s) of the dsRNA agent. In other embodiments, the carrier replaces the nucleotides at the terminal end of the sense 20 strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide on the 3’ end of the sense strand, thereby functioning as an end cap protecting the 3’ end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for instance, the carrier may be 110 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • a ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is 5 referred to herein as a ribose replacement modification subunit (RRMS).
  • the carrier can be a cyclic or acyclic moiety and include two “backbone attachment points” (e.g., hydroxyl groups) and a ligand (e.g., the lipophilic moiety).
  • the one or more C22 hydrocarbon chains can be directly attached to the carrier or indirectly attached to the carrier by an intervening linker/tether, as described above.
  • the ligand-conjugated monomer subunit may be the 5’ or 3’ terminal subunit of the iRNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides.
  • the ligand-conjugated monomer subunit may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be 15 present in an iRNA agent.
  • Cyclic sugar replacement-based monomers e.g., sugar replacement-based ligand-conjugated monomers
  • the carriers may have the 20 general formula (LCM-2) provided below (in that structure preferred backbone attachment points can be chosen from R 1 or R 2 ; R 3 or R 4 ; or R 9 and R 10 if Y is CR 9 R 10 (two positions are chosen to give two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 )).
  • Preferred tethering attachment points include R 7 ; R 5 or R 6 when X is CH 2 .
  • the carriers are described below as an entity, which can be incorporated into a strand.
  • the structures also encompass the situations 25 wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R 1 or R 2 ; R 3 or R 4 ; or R 9 or R 10 (when Y is CR 9 R 10 ), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone.
  • one of the above-named R groups can be -CH 2 -, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.
  • 111 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • X is N(CO)R 7 , NR 7 or CH 2 ; 5 Y is NR 8 , O, S, CR 9 R 10 ; Z is CR 11 R 12 or absent;
  • R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , or (CH 2 ) n OR b , provided that at least two of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 are OR a and/or (CH 2 ) n OR b ;
  • Each of R 5 , R 6 , R 11 , and R 12 is, independently, a ligand, H, C 1 -C 6 alkyl optionally substituted 10 with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 ;
  • R 7 can be a
  • R b is P(O)(O-)H, P(OR 15 )N(R 16 ) 2 or L-R 17 ; 25 R c is H or C 1 -C 6 alkyl; R d is H or a ligand; Each Ar is, independently, C 6 -C 10 aryl optionally substituted with C 1 -C 4 alkoxy; n is 1-4; and q is 0-4.
  • the carrier may be based on the pyrroline ring system or the 4- 35 hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent (D).
  • OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (- CH2OFG 1 in D).
  • OFG 2 is preferably attached directly to one of the carbons in the five-membered ring (-OFG 2 in D).
  • -CH2OFG 1 may be attached to C-2 and OFG 2 may be 5 attached to C-3; or -CH2OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4.
  • CH2OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • -CH2OFG 1 may be attached to C-2 and OFG 2 may be attached to C-4.
  • the pyrroline- and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage,10 e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • CH2OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • the 15 centers bearing CH2OFG 1 and OFG 2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa).
  • the tethering attachment point is preferably nitrogen.
  • Preferred examples of carrier D include the following: 20 . 113 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO I n certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is .
  • OFG 2 is preferably attached directly to one of the carbons in the six- membered ring (-OFG 2 in E).
  • -(CH2)nOFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4.
  • - (CH2)nOFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be 10 attached to adjacent ring carbon atoms, e.g., -(CH2)nOFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH2)nOFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; - (CH2)nOFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH2)nOFG 1 may be attached to C-4 and OFG 2 may be attached to C-3.
  • the piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular 15 linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH2)nOFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • the carriers may be based on the piperazine ring system (F), e.g., X is r the morpholine ring system (G), e.g., X is N(CO)R7 25 .
  • OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (-CH 2 OFG 1 in F or G).
  • OFG 2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG 2 in F or 114 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO G).
  • -CH2OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or vice versa.
  • CH2OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • the piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, 5 e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • CH2OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures.
  • R can be, e.g., C1-C6 alkyl, preferably CH3.
  • the tethering attachment point is preferably nitrogen in both F and G.
  • OFG 2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG 2 in H).
  • -(CH2)nOFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5.
  • -(CH2)nOFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., - 25 (CH2)nOFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH2)nOFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; -(CH2)nOFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH2)nOFG 1 may be attached to C-4 and OFG 2 may be attached to C-3; - (CH2)nOFG 1 may be attached to C-4 and OFG 2 may be attached to C-5; or -(CH2)nOFG 1 may be attached to C-5 and OFG 2 may be attached to C-4.
  • the decalin or indane-based monomers may 30 therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH2)nOFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual 35 diastereomers and diastereomeric mixtures.
  • the monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual 10 diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG 1 and OFG 2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa).
  • the tethering attachment point is preferably nitrogen. Details about more representative cyclic, sugar replacement-based carriers can be found in 15 U.S.
  • Acyclic sugar replacement-based monomers e.g., sugar replacement-based ligand-conjugated 20 monomers, are also referred to herein as ribose replacement monomer subunit (RRMS) monomer compounds.
  • Preferred acyclic carriers can have formula LCM-3 or LCM-4: .
  • each of x, y, and z can be, independently of one another, 0, 1, 2, or 3.
  • I n formula LCM-3, when y and z are different, then the tertiary carbon can have either the R or S 25 configuration.
  • x is zero and y and z are each 1 in formula LCM-3 (e.g., based on serinol), and y and z are each 1 in formula LCM-3.
  • formula LCM-3 or LCM-4 below can optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl. Details about more representative acyclic, sugar replacement-based carriers can be found in U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their 30 entireties. 116 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, e.g., a sense strand or an antisense strand.
  • Internal positions of a strand refers to the nucleotide on any position of the strand, except the terminal position from the 3’ end and 5’ end of the strand (e.g., excluding 2 positions: position 1 counting from the 3’ end and 5 position 1 counting from the 5’ end).
  • the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, which include all positions except the terminal two positions from each end of the strand (e.g., excluding 4 positions: positions 1 and 2 counting from the 3’ end and positions 1 and 2 counting from the 5’ end).
  • the at least one in 10 vivo delivery enhancing moiety is conjugated to one or more internal positions on at least one strand, which include all positions except the terminal three positions from each end of the strand (e.g., excluding 6 positions: positions 1, 2, and 3 counting from the 3’ end and positions 1, 2, and 3 counting from the 5’ end).
  • the at least one in vivo delivery enhancing moiety may be conjugated to 15 one or more internal positions on at least one strand, except the cleavage site region of the sense strand, for instance, the at least one in vivo delivery enhancing moiety is not conjugated to positions 9- 12 counting from the 5’-end of the sense strand, for example, the at least one in vivo delivery enhancing moiety is not conjugated to positions 9-11 counting from the 5’-end of the sense strand.
  • the internal positions exclude positions 11-13 counting from the 3’-end of the sense 20 strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, which exclude the cleavage site region of the antisense strand. For instance, the internal positions exclude positions 12-14 counting from the 5’-end of the antisense strand. 25 In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one or more internal positions on at least one strand, which exclude positions 11-13 on the sense strand, counting from the 3’-end, and positions 12-14 on the antisense strand, counting from the 5’- end.
  • the at least one in vivo delivery enhancing moiety may be conjugated to 30 one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5’end of each strand. In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’end of each strand. 35 In one embodiment, the at least one in vivo delivery enhancing moiety may be conjugated to position 6 on the sense strand, counting from the 5’end of each strand.
  • the at least one in vivo delivery enhancing moiety may be conjugated to a nucleobase, sugar moiety, or internucleosidic phosphate linkage of the dsRNA agent.
  • the at least one in vivo delivery enhancing moiety is conjugated to a sugar moiety of the dsRNA agent.
  • the at least one in vivo delivery enhancing moiety is conjugated to the 2’ position of a ribose sugar of the dsRNA agent. 5 V.
  • a dsRNA agent comprises an antisense strand and a sense strand; at least one ⁇ v ⁇ 6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to at 10 least one strand.
  • S are described in U.S.
  • Integrins are cell surface receptors that, upon ligand binding, activate signal transduction pathways including signaling pathways involved in cytoskeleton organization and cell cycle 20 regulation. Integrins are also involved in cell attachment to the extracellular matrix and the integrin ligands comprise common extracellular matrix components, including fibronectin, collagen, laminin, fibrinogen, thrombospondin, and glycoproteins (e.g., tenascin C, osteopontin, and nefronectin). In humans there are at least twenty-four known integrin heterodimers composed of an alpha and a beta subunit, e.g., ⁇ v ⁇ 6.
  • ⁇ v ⁇ 6 cytokine transforming growth factor-b1
  • LAP pro-peptide latency associated 30 peptide
  • TGF- ⁇ 1 regulates multiple processes including cell proliferation, differentiation, angiogenesis, epithelial-mesenchymal- transition (EMT) and immune suppression. These processes combine to heal wounds but when uncontrolled can promote tissue pathologies.
  • the present disclosure provides dual conjugated dsRNA agents for inhibiting expression of a 35 DMPK gene that comprise an antisense strand and a sense strand; at least one ⁇ v ⁇ 6 integrin targeting ligand that mediates delivery to muscle tissue conjugated to at least one strand; and at least one in vivo delivery enhancing moiety conjugated to at least one strand.
  • 118 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO A .
  • the dsRNA agent of the present disclosure is conjugated to at least one ⁇ v ⁇ 6 integrin targeting ligand.
  • the at least one ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to the dsRNA agent via a direct attachment to the dsRNA agent, e.g., a ribosugar of the dsRNA agent.
  • the at least one ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to the dsRNA agent via a linker or a carrier as described herein.
  • Exemplary ⁇ v ⁇ 6 integrin targeting ligands that can conjugated to the dsRNA agents of the disclosure can be found, e.g., in U.S. Patent Nos 10,023,568, 10,450,312, 10,144,733, 10,487,080, 105,13,517, and 10,000,489, as well as in International Patent Application No. PCT/US2023/083947, 10 filed on December 14, 2023, the entire contents of each of which are incorporated herein by reference.
  • Exemplary ⁇ v ⁇ 6 integrin targeting ligands are also described in WO2024/086633, incorporated herein by reference.
  • the ⁇ v ⁇ 6 integrin targeting ligand may be conjugated to the dsRNA agent via a linker comprising a compound of the structure 15 L333 N-(aminocaproyl-DBCO)-4-hydroxyprolinol, wherein * represents the bond to the remainder of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 20 119 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5 bond from R Lig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 10 wherein R Lig is 120 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO bond to the remainder of the dsRNA agent. In one embodiment, the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 1 121 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO bond from R Lig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the 5 remainder of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 10 122 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5 bond from R Lig to the nitrogen, # is the bond to the integrin ligand, and * represents the bond to the remainder of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 10 wherein R Lig is 123 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO bond to the remainder of the dsRNA agent.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: , 5 wherein * represents the bond to the remainder of the dsRNA agent. In one embodiment, the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: , wherein * represents the bond to the remainder of the dsRNA agent. 10 In one embodiment, the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: , wherein * represents the bond to the remainder of the dsRNA agent. In one embodiment, the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 124 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: 5 , wherein * represents the bond to the remainder of the dsRNA agent. In one embodiment, the ⁇ v ⁇ 6 integrin targeting ligand comprises the structure: , wherein * represents the bond to the remainder of the dsRNA agent. 10 In some embodiments, the ⁇ v ⁇ 6 integrin targeting ligand or the ⁇ v ⁇ 6 integrin targeting ligand to the dsRNA agent of the present disclosure comprises a structure selected from the table below: 125 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO 126 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Z1 is a linker linking the ⁇ v ⁇ 6 integrin targeting ligand to the remainder of the dsRNA agent and wherein * represents the bond to the remainder of the dsRNA agent.
  • Z1 is a linker linking the ⁇ v ⁇ 6 integrin targeting ligand to the remainder of the dsRNA agent and wherein * represents the bond to the remainder of the dsRNA agent.
  • 127 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO B .
  • the alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand may be conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of the disclosure, 5 via a carrier group.
  • the alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand conjugated to the dsRNA agent of the disclosure is represented by a compound of the Formula (X): or a salt thereof, wherein: 10 r CH2; (e.g., O or CH2) , wherein m is 0, 1, 2, 3, or 4; and each R2 is independently R, or two R2 groups on adjacent carbon atoms taken together with the atoms to which they are bound form a fused 4 – 8 membered ring (e.g,.
  • A is an 5-membered heteroaryl optionally substituted with 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl;
  • 20 Q is -COOR1 or tetrazolyl (e.g., 1,2,3,4-tetrazol-5-yl), wherein R1 is hydrogen or C1-6alkyl (e.g., methyl) ; and R L is -N(R 3 )(R 4 ), -O(R 5 ), -S(R 5 ), or -R 5 , wherein R 3 and R 4 are either 25 (i) R3 is hydrogen or C1-6alkyl and R4 is R5; or (ii) R3 and R4 taken together with the nitrogen
  • ALN-529-WO q is 0 or an integer selected from 1 – 25; (e.g., 1-20, or 1-15);
  • L 1 is a bond or -B-A-;
  • each L 2 is independently -A-B-A-;
  • L 3 is a bond or -A-B-A-;
  • 5 each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • each A is independently a bond, -O-, -S-, or -N(R N )-;
  • each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O), S(O)2, P(O)(
  • each -D-E-F- group at least one of D, E, and F is not a bond; and R L is not N-morpholinyl.
  • R 3 and R 4 do not form a morpholino ring. 5
  • B is only a bond when one of the A groups is not a bond.
  • each R group is independently selected from the group consisting of R’, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-8 cycloalkyl, heterocyclyl, aryl, heteroaryl, C 3- 8 cycloalkylC 1-6 alkyl, heterocyclylC 1-6 alkyl, aryl C 1-6 alkyl and heteroaryl 1-6 alkyl, each of which, other 10 than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(R b ) 2 , -O(R a ), -S(R 0 ), -C(O)OR 0 , -C(O)R 0 , -C(O)N(R 0 ) 2 , -N(R 0 )C(O)R 0 ,
  • each R group is independently selected from the group consisting of R’, C 1-6 alkyl, C 1-6 haloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, and benzyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each R’ is independently halogen, cyano, azido, nitro, -N(R b )2, - O(R a ), -S(R 0 ), -C(O)OR 0 , -C(O)R 0 , -C(O)N(R 0 )2, -N(R 0 )C(O)R 0 , -OC(O)OR 0 , or -OC(O)R 0 ,20 wherein each R 0 is independently hydrogen
  • each R group is independently selected from the group consisting of R’, C1-6alkyl, C3-6cycloalkyl, 5-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl, and benzyl, each of which, other than R’, is optionally substituted with 1, 2, or 3 R’ groups, wherein each25 R’ is independently halogen, cyano, azido, nitro, -N(R b )2, -O(R a ), -S(R 0 ), -C(O)OR 0 , - C(O)R 0 , -C(O)N(R 0 )2, -N(R 0 )C(O)R 0 , -OC(O)OR 0 , or -OC(O)R 0 , wherein each R 0 is independently hydrogen or C1-6alkyl; each R a is independently hydrogen or C1-6alkyl; and each R b is independently hydrogen, C
  • the compound of formula ( 30 In certain embodiments, the compound of formula ( 130 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO In certain embodiments, the compound of formula ( In certain embodiments, the compound of formula ( 5 A.
  • Formula (X) is selected from an optionally substituted N- or a C- linked pyrazole, an optionally substituted N- or a C-linked triazole, and an optionally substituted N- or C-linked imidazole, wherein the optionally substituted N- or a C-linked pyrazole, the optionally substituted N- or a C-linked triazole, and the optionally substituted N- or C-linked imidazole is 10 optionally substituted by 1 or 2 substituents independently selected from methyl, ethyl, fluoro, hydroxymethyl, 2-hydroxypropan-2-yl, trifluoromethyl, difluoromethyl, and fluoromethyl.
  • R1 is hydrogen, methyl, or ethyl
  • R2 is hydrogen or fluoro
  • R3 is hydrogen, methyl, or ethyl.
  • R1 is hydrogen, methyl, or ethyl
  • R2 is hydrogen or fluoro
  • R3 is hydrogen, methyl, or ethyl.
  • Formulae (X) In some embodiments of any one of Formula (X), in R Y , each R 2 is independently R as defined in Formula (X). 5
  • R Y is (or its tautomer, ). In some embodiments, .
  • R Y two R 2 groups on adjacent carbon atoms taken together with the atoms 10 to which they are bound form a fused 4 – 8 membered ring that is optionally substituted with 1, 2, 3 or 4 groups independently selected from group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV).
  • R is as defined in Formula (IV).
  • 15 4; and each R 21 is independently selected from group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV).
  • R Y is , wherein p is 0, 1, 2, 3 or 4; and each R 21 is independently selected from the group consisting of R and a nitrogen protecting group, wherein R is as defined in Formula (IV).
  • R P is , wherein r is 0, 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C 1- 4 alkoxy, C 1-4 alkyl, C 1-4 haloalkyl; and each R P3 is independently hydrogen, methyl, or ethyl.
  • R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2- 5 chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4-dichlorophenoxyacetyl, 2- methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4-chloro-2- methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t- butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-10 fluorophenoxyacetyl,
  • R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2- chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4-methylphenoxyacetyl, or 4- 15 isopropylphenoxyacetyl.
  • R P is methoxyacetyl (mac).
  • R P is phenoxyacetyl (pac).
  • R 1 is C1-6alkyl , wherein 20 r is 0, 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C1-4alkoxy, C1-4alkyl, C1-4haloalkyl; and each R P3 is independently hydrogen, methyl, or ethyl.
  • R 1 is C1-6alkyl and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4-dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 25 4-chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2-isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t- butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl,
  • R 1 is C 1-6 alkyl and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4- methylphenoxyacetyl, or 4-isopropylphenoxyacetyl.
  • R 1 is C 1-6 alkyl and R P is methoxyacetyl (mac).
  • R 1 when present, R 1 is C 1-6 alkyl and R P is phenoxyacetyl (pac). In some embodiments, when present, R 1 is methyl , wherein r is 0, 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C 1-4 alkoxy, C 1-4 alkyl, C 1-4 haloalkyl; and each R P3 is independently hydrogen, methyl, or ethyl.
  • R 1 is methyl and R P is methoxyacetyl (mac),10 phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4- chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2- isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t- butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3-15 fluorophenoxyacet
  • R 1 when present, R 1 is methyl and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4- 20 methylphenoxyacetyl, or 4-isopropylphenoxyacetyl.
  • R 1 when present, R 1 is methyl and R P is methoxyacetyl (mac). In some embodiments, when present, R 1 is methyl and R P is phenoxyacetyl (pac).
  • R 1 is hydrogen , wherein r is 0, 1, 2, or 3; each R P2 is independently halogen, nitro, cyano, C1-4alkoxy, C1-4alkyl, C1-4haloalkyl; and 25 each R P3 is independently hydrogen, methyl, or ethyl.
  • R 1 is hydrogen and R P is methoxyacetyl (mac), phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 3-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2,4- dichlorophenoxyacetyl, 2-methylphenoxyacetyl, 3-methylphenoxyacetyl, 4-methylphenoxyacetyl, 4- chloro-2-methylphenoxyacetyl, 2-nitrophenoxyacetyl, 3-nitrophenoxyacetyl, 4-nitrophenoxyacetyl, 2-30 isopropylphenoxyacetyl, 3-isopropylphenoxyacetyl, 4-isopropylphenoxyacetyl, 2-(t- butyl)phenoxyacetyl, 3-(t-butyl)phenoxyacetyl, 4-(t-butyl)phenoxyacetyl, 2-fluorophenoxyacetyl, 3- 135 ME1 ⁇ 53453057
  • R 1 when present, R 1 is hydrogen and R P is methoxyacetyl (mac), 5 phenoxyacetyl (pac), 2-chlorophenoxyacetyl, 4-chlorophenoxyacetyl, 2-methylphenoxyacetyl, 4- methylphenoxyacetyl, or 4-isopropylphenoxyacetyl.
  • R 1 when present, R 1 is hydrogen and R P is methoxyacetyl (mac).
  • R 1 when present, R 1 is hydrogen and R P is phenoxyacetyl (pac). 10 R T Embodiments, Formula (X) In some embodiments of Formula (X), R T is R T1 .
  • R T is -G 0 -OR T1 , wherein R T1 is as defined for Formula (X) and G 0 is selected from: ( a) G0 is absent or -D0-E0-F0-, wherein D0, E0, and F0 are independently a bond, C1- 15 10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; ( b) G0 is -D0-E0-F0-, wherein D 0 and F 0 are independently a bond or C 1-10 alkyl optionally substituted with 1, 2, 3, or 4 R groups; and 20 E 0 is C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; ( a) G0 is absent or
  • R groups e.g., 1 or 2 R groups
  • G0 is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups
  • h G0 is C1-10alkyl optionally substituted with 1 or 2 R groups
  • 10 (i) G0 is C1-10alkyl, optionally substituted with -O(Ra), wherein Ra is independently hydrogen or C1-6alkyl
  • j G0 is C1-10alkyl
  • G0 is absent (a bond); wherein * represents the bond to L’, the broken bond represents the bond to OR T1 , and R is - 15 C1-6alkyl-OR a or -OR a , wherein R a is independently hydrogen or C1-6alkyl.
  • Formula (X) I n some embodiments of Formula (X), -L’- is *-G-L1-, wherein * is the bond to ZZ; and (a) L1 is a bond or -B-A-, wherein 20 A is a bond, -O-, -S-, or -N(R N )-; B is a bond, CH2, C(O), C(S), C(NR N ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C1-6alkyl; and G is -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is 25 optionally substituted with 1, 2, 3, or
  • ALN-529-WO G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • ( c) L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, 5 or 4 R groups;
  • ( d) L1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and
  • G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; or 10 (e
  • -L’- is *-G-[L2-G]q-L1-, wherein * is the bond to ZZ; q, is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer selected from 1 to 8, an integer selected from 1 to 5, or an integer from 1 to 3); and ( a) L1 is a bond or -B-A-; 15 each L 2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C1-6alkyl; 20 each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkeny
  • ( c) L1 is a bond, CH2, C(O), C(S), C(NRN), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each L 2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein R N is hydrogen or C1-6alkyl 35 each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups ( d) L1 is a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH) each L 2 is independently -A-B-A-; 138 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Aln
  • each A is independently a bond, -O-, -S-, or -N(R N )-, wherein R N is hydrogen or C1-6alkyl each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl, optionally substituted with 1, 2, 3, or 4 R groups; or 5 (e) L1 is a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); each L 2 is independently -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein R N is hydrogen or C1-6alkyl each B is independently a bond, CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); each G is independently C1-10alkyl; 10 In some embodiment
  • each L 2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; or 5 (vii) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L 2 is a bond; each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is *-L 3 -G-L 1 -, wherein * is the bond to ZZ; and ( a) L1 and L3 are independently -A-B-A-; 10 each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein R N is hydrogen or C1-6alkyl; and each B is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); 15 (b) L3 is -C(O)O- or C(O)N(RN)-, wherein RN is hydrogen or C1-6alkyl; L 1 is -OP(O)
  • -L’- is *-G-, wherein * is the bond to ZZ; and G is C1-10alkyl is optionally substituted with 1 or 2 R groups.
  • -L’- is --L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to ZZ;
  • -L’- is -L 1 -G-L 3 -*, wherein * is the bond to ZZ;
  • 35 In some embodiments, -L’- is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, or 5; 140 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO L 1 is a bond or -B-A-; each L 2 is independently -A-B-A-; L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; 5 each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each R N is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • -L’- is -L 1 -[G-L 2 ]q-G-*, wherein * is the bond to ZZ; q is 0, 1, 2, or 3; L 1 is a bond or -B-A-; each L 2 is independently a bond, C(O)O, OC(O), C(O)(NR N ), N(R N )C(O), SO2N(R N ), N(R N )SO2, OP(O)(OH), OP(S)(OH), P(O)(OH)O, P(S)(OH)O, OP(O)(OH)O, or OP(S)(OH)O, wherein 15 each R N is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is -L 1 -[G-L 2 ]q-G-*, wherein * is the bond to ZZ; q is 0, 1, 2, or 3; 20 L 1 is a bond or -B-A-; each L 2 is independently a bond, C(O)O, OC(O), C(O)(NR N ), N(R N )C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each R N is independently hydrogen or C1-6alkyl; each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is -[G-L 2 ]q-G-*, wherein * is the bond to ZZ; and ( a) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each L 2 is independently C(O)O or OC(O); each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; ( b) q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); 30 each L 2 is independently C(O)(NR N ) or N(R N )C(O), wherein each R N is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups; ( c) q is 0, 1, 2, or 3 (e.g., q is 0, 1,
  • each L 2 is a bond; and each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is -C2-30alkyl-*, wherein * is the bond to ZZ, such as -C5- 20alkyl-* or -C10-20alkyl-*.
  • -L’- is -C(O)-C2-30alkyl-*, wherein * is the bond to ZZ, such as -C(O)- C5-20alkyl-* or -C(O)-C10-20alkyl-*.
  • L’-R T Embodiments In some embodiments, - , wherein 10 * is the bond to ZZ; L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and R T1 is as defined for Formula (X). In some embod 15 wherein * is the bond to ZZ; L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); and R T1 is as defined for Formula (X).
  • L 1 is a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); R is -OH or -C 1-6 alkyl-OH; and R T1 is as defined for Formula (X).
  • * is the bond to ZZ; L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); 142 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO R is -OH or -C1-6alkyl-OH; and R T1 is as defined for Formula (X).
  • each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,- OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; or ( ii) independently selected from the group consisting of -C(O)O-, -
  • -L’-R T is 143 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein * is the bond to ZZ; 5 q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,- 10 OP(S)(OH)O-, -O-, and -N(R
  • -L’-R T is , , or , wherein * is the bond to ZZ; 30 q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); each L 2 is 144 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,- OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; or ( ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, 5 -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N
  • each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,- 25 OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl; or ( ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-
  • ALN-529-WO ( vi) independently selected from the group consisting of -C(O)N(RN)- and -N(RN)C(O), wherein each R N is independently hydrogen or C1-6alkyl; L 1 is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); R is -OH or -C1-6alkyl-OH; and R T1 is as defined for Formula (X).
  • each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,- OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; or 15 (ii) independently selected from the group consisting of -C(O)O-,
  • -L’-R T is 146 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein* is the bond to ZZ; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, or an integer from 1 to 3); 5 each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,- OP(S)(OH)O-, -O-, and -N(R
  • each L 2 is ( i) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,- OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; or 5 (ii) independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(RN)-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )-,
  • R is -O(R a ) or -C 1-6 alkyl-O(R a ), wherein R a is hydrogen; and 25 R T1 is as defined for Formula (X).
  • R L Embodiments, Formula (X)
  • R L is -N(R 3 )(R 4 ), -O(R 5 ), -S(R 5 ), or -R 5 , wherein R 5 is according to one Formulae (x-a) through (x-ab): ( x-a) (x-b) 148 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 149 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • R P3 is hydrogen and L, ZZ, L’ and R T1 are as defined for Formula (X) or any embodiment herein.
  • R L is according to one of Formulae (xi-a) through (xi-m): 150 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO (xi-k) (xi-l) 151 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein R P3 is hydrogen, and L, ZZ, L’ and R T1 are as defined for Formula (X) or any embodiment herein.
  • R L is according to one of Formulae (xi-n) through (xi-z): (xi-w) (xi-x) (xi-y) 152 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO (xi-z) wherein R P3 is hydrogen, and L, ZZ, L’ and R T1 are as defined for Formula (X) or any embodiment herein.
  • R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen or C1-6alkyl and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen and R 4 is R 5 . 5 In some embodiments, R L is -N(R 3 )(R 4 ), wherein R 3 is C1-3alkyl and R 4 is R 5 . In some embodiments, R L is -N(R 3 )(R 4 ), wherein R 3 is methyl and R 4 is R 5 . In some embodiments, R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 .
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 , provided that R 3 and R 4 taken together with the nitrogen atom to which they are attached do not form a morpholino group.
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen 15 atom to which they are attached form a group that is piperidinyl, piperazinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, azetidinyl, pyrrolinyl, imidazolinyl, or pyrazolinyl, each substituted with R 5 .
  • R L is -N(R 3 )(R 4 ).
  • R L is -N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 .
  • R L is -O(R 5 ).
  • R L is -R 5 .
  • 24 e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • 153 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • RL is , wherein * is a bond to a ZZ group; t is 0 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3).
  • L Embodiments, Formula (X) In some embodiments of any one of Formula (X) and any embodiments thereof, L is -L 1 -[G- 5 L 2 ] q -G-L 3 -*, wherein * is the bond to ZZ. In another embodiment, wherein L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, 2, 3, or 4
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, 2, or 3.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 0, 1, or 2.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 1, 2, 3, or 4.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 1, 2, or 3.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 1 or 2.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 4.
  • L 15 is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 3.
  • L is -L 1 -[G-L 2 ] q -G-L 3 -*, wherein q is 2.
  • L is -L 1 -G-L 2 -G-L 3 -*.
  • L is -L 1 -G-L 3 -*.
  • L is -G-L 3 -*.
  • L is -L 1 -G-*.
  • L is -G-*.
  • L is -[G-L 2 ] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or 20 q is 3; or q is 4; or q is 5).
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, - N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-,- N(R N )C(O)N(R N )-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -O-, 30 and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • each instance of A-B-A- is independently selected from the group 35 consisting of -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C 1-6 alkyl.
  • 154 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO D and F are each independently a bond, C1-10alkyl, C2-10alkenyl, or C2-10alkynyl, each optionally substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • D and F are each independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, optionally substituted with 1, 2, or 3 R groups. In some embodiments, each G is independently C 1-10 alkyl. 15 In some embodiments, each G is independently C1-10alkyl, optionally substituted with 1 or 2 R groups. In some embodiments, each G is independently C1-10alkyl, optionally substituted with one R group..
  • L is -L 1 -G-L 3 -*, wherein * is the bond to ZZ ;
  • 20 G is -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-A-;
  • L 3 is a bond or -A-B-A-;
  • 25 each A is independently a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and
  • L is -L 1 -G-L 3 -*, wherein * is the bond to ZZ;
  • G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, 30 or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-;
  • L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; and 35 each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH; and
  • L is -L 1
  • ALN-529-WO L 1 is -B-A-, wherein, A is a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; and B is a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to ZZ;
  • G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1 or 2 R groups;
  • L 1 is -B-A-, wherein A is a bond, -O-, -S-, or -N(R N )-, each R N is independently hydrogen or C1-6alkyl; and 10 B is a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to ZZ; G is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; L 1 is bond, C(O), S(O)2, P(O)(OH), or P(S)(OH); and R N is hydrogen or C1-6alkyl.
  • L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L 1 is bond, C(O), C(S), C(NR N ), S(O) 2 , P(O)(OH), or P(S)(OH); and R N is hydrogen or C 1- 6 alkyl.
  • L is wherein * is the bond to ZZ; k is an integer from 1 to 10; L 1 is bond, C(O), P(O) 20 In some embodiments, wherein * is the bond to ZZ; k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7.
  • each G is independently C 1-10 alkyl and L 2 is O, S, or N(H). 25
  • L is -[G-L 2 ] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5)
  • each G is independently C 1-10 alkyl and L 2 is O.
  • L is -[CH 2 CH 2 O] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5), G is independently C 1-10 alkyl.
  • L is , wherein * is the bond to ZZ; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3).
  • I n some embodiments, L is , wherein * is the bond to ZZ. 156 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • L is , wherein * is the bond to ZZ; t is 1 to 10 (e.g., 1-5; or 1-3; or 1; or 2; or 3); and z is 1-10 (e.g., 1-6; or 1-4; or 1; or 2; or 3 or 4 or 5 or 6).
  • * is the bond to ZZ, and t is an integer from 1 to 5 (e.g., 1; or 2; or 3).
  • t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an integer from 3 to 7; or an 10 integer from 4 to 6).
  • * is the bond to ZZ; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ; and s, s’, 15 and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • * is the bond to ZZ and each s, s’, and s” independently is an integer from 1 to 24(e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6) 20
  • L is wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is C1-20 alkyl.
  • L is C2-20 alkyl.
  • L is C6-20 alkyl.
  • L is C8-12 alkyl.
  • L is C10 alkyl.
  • RP3 when present, is hydrogen.
  • the compound of Formula (X) is according to one of Formulae (X-b) 10 through (X-e) and (X-x) through (X-y): 158 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO or a salt thereof, wherein R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), wherein R and the remaining variables are as defined in Formula (X).
  • R 1 is hydrogen. In another embodiment of Formulae (X-b) through (X-e) and (X-x) through (X-y), R 1 is C 1-6 alkyl (e.g., methyl or 5 t-butyl). In another embodiment of Formulae (X-b) through (X-e) and (X-x) through (X-y), R 1 is hydrogen and R P is hydrogen. In another embodiment of Formulae (X-b) through (X-e) and (X-x) through (X-y), R 1 is hydrogen and a nitrogen protecting group.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., 10 methyl or t-butyl) and a nitrogen protecting group.
  • -L-ZZ-L’-R T Embodiments
  • -L-ZZ-L’-R T is 159 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO (x-a).
  • -L-ZZ-L’-R T is (x-b).
  • -L-ZZ-L’-R T is (x-c). 5
  • -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) 10 through (X-y), -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formula
  • -L-ZZ-L’-R T is (x-h). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is (x-i). 5 In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is (x-j).
  • -L-ZZ-L’-R T is (x-k). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) 10 through (X-y), -L-ZZ-L’-R T is (x-l). 161 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • -L-ZZ-L’-R T is (x-m). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is (x-n). 5 In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is (x-o).
  • -L-ZZ-L’-R T is In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is (x-q). In an embodiment, of any one of Formula (X) and Formulae (X-a) through (X-e) and (X-x) through (X-y), -L-ZZ-L’-R T is 162 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 3’-end of the 10 oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on an internal nucleotide (i.e., a nucleotide that is not the 5’-terminus
  • L L is a bond.
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 3’-end of the oligonucleotide, such as one of: 15 directly to the 3’-carbon of the 3’-terminal nucleoside; directly to the 3’-O of the 3’-terminal nucleoside; directly to the 4’-carbon of the 3’-terminal nucleoside; directly to the 2’-carbon of the 3’-terminal nucleoside; or directly to the 2’-O of the 3’-terminal nucleoside.
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; directly to the 4’-carbon of the 5’-terminal nucleoside; 25 directly to the 2’-carbon of the 5’-terminal nucleoside; or directly to the 2’-O of the 5’-terminal nucleoside.
  • L L is a divalent linker that connects to the 5'-end of the oligonucleotide, such as one of: directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; directly to the 4’-carbon of the 5’-terminal nucleoside; 25 directly to the 2’-carbon of the
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internal 2’- or 3’ position on an internal nucleotide.
  • L L is a divalent linker that connects to an internal 2’- or 3’ position on an internal nucleotide.
  • R T is R T1 , wherein R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internucleotide linkage (i.e., to an oxygen atom in a phosphodiester linkage 5 to form a phosphotriester; or to a nitrogen when the internucleotide linkage is a phosphoroamidate).
  • L L is a divalent linker that connects to an internucleotide linkage (i.e., to an oxygen atom in a phosphodiester linkage 5 to form a phosphotriester; or to a nitrogen when the internucleotide linkage is a phosphoroamidate).
  • L L when L L connects to a carbon atom on a nucleoside, then L L is -B 3 -A 3 -, wherein B 3 is -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH)-; and A 3 is -O-, -S-, or -N(H)- . 10
  • L L when L L connects to a carbon atom on a nucleoside, then L L is -B 3 - A 3 -, wherein B 3 is -P(O)(OH)- or-P(S)(OH)-; and A 3 is -O-.
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is - 15 P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH). In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)- or -P(S)(OH)-. In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)-. 20 In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(S)(OH)-.
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is -B3-A3-LL1-A3-B3-, wherein B 3 is a bond, -C(O)-, C(S)-, C(NH), S(O), S(O)2, -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH); 25 A 3 is a bond, -O-, -S-, or -N(H)- ; and L L1 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl.
  • L L when LL connects to a oxygen atom on a nucleoside, then LL is -B3- L L1 -B 3 -, wherein B 3 is -C(O)-; and L L1 is C1-10alkyl. 30 In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is-a bond.
  • R T is R T1 wherein R T1 is is -L L -oligonucleotide, when L L connects to a oxygen atom on a nucleoside, and L L is a bond, the nucleoside is of Formula (X-f), 164 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); L’ is according any of the preceding embodiments; and * represent the bond to ZZ.
  • R T1 is is -L L -oligonucleotide, when L L connects to a oxygen atom on a nucleoside, and L L is a bond, the nucleoside is of Formula (X-f), 164 ME1 ⁇ 53453057.v1 Attorney Docke
  • -L’- is -L 1 -G-L 3 -*, wherein * is the bond to ZZ. 5
  • -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two R groups, and * is the bond to ZZ.
  • -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two groups selected from the group consisting of halogen, hydroxy, C1-6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group 10 selected from the group consisting of halogen, hydroxy, C1-6alkoxy, amino, Cl-6alkylamino, di(C1- 6alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from the group consisting of hydroxy, amino, and carboxy , and * is the bond to ZZ.
  • -L’- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with hydroxy, and * is the bond to ZZ.
  • -L’- is -C2-30alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -C2-16alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -C4-12alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -C4-10alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -C5-10alkyl-*, wherein * is the bond to ZZ. In some embodiments, -L’- is -C6alkyl- *, wherein * is the bond to ZZ. In some embodiments, -L’- is -C8alkyl-*, wherein * is the bond to ZZ. 20 In some embodiments, -L’- is -C10alkyl-*, wherein * is the bond to ZZ.
  • -L’- is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, or 5; L 1 is a bond or -B-A-; 25 each L 2 is independently -A-B-A-; L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O)2, P(O)(OH), or P(S)(OH); each R N is independently hydrogen or C1-6alkyl; and 30 each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • -L’- is -L 1 -[G-L 2 ]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3;
  • each L2 is independently a bond, C(O)O, OC(O), C(O)(NRN), N(RN)C(O), OP(O)(OH)O, or OP(S)(OH)O, wherein each R N is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups.
  • -L’- is -[G-L 2 ]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3 (e.g., 5 q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each G is independently C1-10alkyl, each of which is optionally substituted with 1 or 2 R groups, and one of: ( a) each L2 is independently C(O)O or OC(O); (b) each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl 10 (c) each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); or ( d) each L2 is a bond.
  • the compound of Formula (X) is according to one of Formulae (X-g) through (X-q): 166 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); 5 each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6).
  • nucleobase e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil
  • each n is independently 0 or an integer selected from 1-10
  • the compound of Formula (X) is according to one of Formulae (X-r) through (X-w): 167 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5 L and ZZ are as defined in Formula (X) or in any embodiment preceding or below;
  • B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); 10 each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6); each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group); and R 1 is hydrogen or C 1-6 alkyl (e
  • R 1 is hydrogen. In another embodiment of Formulae (X-r) through (X-w), R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In another embodiment of Formulae (X-r) through (X-w), R 1 is hydrogen and R P is hydrogen. In another embodiment of Formulae (X-r) through (X-w), R 1 is hydrogen and a nitrogen protecting group.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P 168 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group.
  • two adjacent nucleosides in the oligonucleotide have one of the 5 oligonucleotide, L’ is according any of the preceding embodiments; and B is an optionally modified nucleobase.
  • each nucleoside of the two adjacent nucleosides is independently according to 10 any one of Formula (X-f) through (X-q).
  • each nucleoside is according to the same Formula.
  • three adjacent nucleosides in the oligonucleotide have the formula 169 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each Y is independently O or S (or O; or S; or O then S or S then O, 5’ followed by 3’); represents the remainder for the oligonucleotide, L’ is according any of the preceding 5 embodiments; and B is an optionally modified nucleobase; e.g., each Y is O.
  • each nucleoside of the three adjacent nucleosides is independently according to any one of Formula (X-f) through (X- q). In certain embodiments, each nucleoside is according to the same Formula.
  • each nucleoside of the four adjacent nucleosides is independently according to any one of Formula (X-f) through (X-q).
  • each nucleoside is according to the same Formula.
  • each nucleoside is according to the same Formula (X-u) or (X-w).
  • R T is R T1 , wherein R T1 is -L L -oligonucleotide, and when L L connects to an oxygen atom or nitrogen atom in an internucleotide linkage, the internucleotide linkage can be of the formula, including the 3’ and 5’ oxygen atoms of the preceding and following nucelosides, respectively, (X-pa) (X-pb) (X-pc) wherein L’ can be, for example a bond, -S(O)2- or, in for Formula (X-pc), a 5 -8 membered 10 heterocyclyl ring optionally substituted with 1 or 2 R groups, as defined herein; and * represent the bond to ZZ.
  • the preceding includes, (X-pd) (X-pe) (X-pf) wherein * represent the bond to ZZ; and R N5 is hydrogen or C1-10 alkyl.
  • the preceding includes, (X-pg) (X-ph) (X-pi) 15 wherein * represent the bond to ZZ; m is an integer selected from 1 – 20 (e.g., 1-10, or 2-20, or 2-10, or 4-10, or 4-8; or 6-12; or 5; or 6; or 7; or 8; or 9; or 10), and R N5 is hydrogen or C1-10 alkyl.
  • the compound of Formula (X-pd) is 171 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R Y , Y, R 1 , L, L’, and ZZ are as defined for Formula (X).
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • the compound is 5 172 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and R 1 , L, L’, and ZZ are as defined for Formula (X). 10
  • the compound of Formula (X-pe) is 174 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • Y’ is O or S, and R Y , Y, R 1 , L, L’, and ZZ are as defined for Formula (X).
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- 5 butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, 10 Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen .
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 20 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 20 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a 25 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound is 175 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5 176 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO w herein Y’ is O or S, and RP, R1, L, L’, and ZZ are as defined for Formula (X). In one embodiment, Y’ is O. In another embodiment, Y’ is S. In one embodiment, R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., methyl or t- butyl). 5 In one embodiment, R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). 10
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl 20 (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 25 (pac)).
  • t-butoxycarbonyl Boc
  • benzyloxycarbonyl Cbz
  • phenoxyacetyl 25 pac
  • ALN-529-WO 5 wherein each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), Y’ is O or S, and R P , R 1 , L, L’, and ZZ are as defined for Formula (X). 178 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group 5 (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a 10 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, 15 R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl 20 or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 5’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-5’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (X).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 5’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-5’o) or Formula (X-5’s), wherein * represents the bond to remainder of the compound of Formula (X). 179 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO In an embodiment of Formula (X-5’), (X-5’o), and Formula (X-5’s), -L-ZZ-L’-R T represents any one of Formulae (x-a) through (x-s) .
  • -L-ZZ-L’-R T represents Formula ( In other embodiments-L-ZZ-L’-R T represents one of the following formulae: ( x-n) (x-o) (x-s) 5 In other embodiments of Formula (X-5’), (X-5’o), and Formula (X-5’s), L-ZZ-L’-R T represents, (x-m) In other embodiments of Formula (X-5’), (X-5’o), and Formula (X-5’s), L-ZZ-L’-R T 10 represents, (x-s).
  • the compound of Formula (X) is 180 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 3’-end of the 5 oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-3’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (X).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at 10 the 3’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (X-3’o) or Formula (X-3’s), wherein * represents the bond to remainder of the compound of Formula (X).
  • -L-ZZ-L’-R T represents 15 any one of Formulae (x-a) through (x-ab).
  • -L-ZZ-L’-R T represents Formula (x-e), .
  • -L-ZZ-L’-R T represents one of the following formulae: ( x-a) (x-b) (x-c) 181 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • L-ZZ-L’-R T represents, In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-R T 5 represents, 182 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO In other embodiments of Formula (X-3’), (X-3’o), and Formula (X-3’s), L-ZZ-L’-R T represents, (x-p).
  • L-ZZ-L’-R T 5 represents, (x-ab).
  • L-ZZ-L’-R T represents, (x-r).
  • R L is 10
  • the compound of Formula (X) is 183 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y' is O or S, and R Y , R 1 , Y, L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., 5 methyl or t-butyl).
  • the compound of Formula (X) is 184 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R Y , Y, R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof. In another embodiment, the compound of Formula (X) is 5 wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O. In another embodiment, Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., methyl or t- 10 butyl). In another embodiment, -COOR1 is replaced with a carboxylic acid mimic, such as a tetrazolyl group (e.g., 1,2,3,4-tetrazol-5-yl).
  • R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 15 6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen .
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a 20 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a 20 nitrogen protecting group
  • e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac) 185 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 5 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a 10 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 15 wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a 5 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, 10 R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl 15 or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 187 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO , wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 5
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1- 10 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or 25 phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 188 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each m is independently an integer selected from 1-10; each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and R P and R 1 are 5 as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl). 10
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C 1- 6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • 189 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 5 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a 10 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 15 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group 20 (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 10 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is 15 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 191 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- 5 butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • 10 Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 20 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a 25 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 192 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof. 5 In one embodiment, Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 193 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen . In one embodiment, Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) 5 or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) 5 or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 10 , wherein each m is independently an integer selected from 1-10; each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); Y’ is O or S, and RP and R1 are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). 194 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • Y’ is O and R P is hydrogen .
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 5 (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 10 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P 15 is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 20 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 195 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 5 In one embodiment, Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 10 (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen 15 protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxy
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) 20 and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 25 , wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., methyl or t- butyl). In one embodiment, R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 5 In one embodiment, Y’ is O and R 1 is hydrogen. In one embodiment, Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 10 (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl 10
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t- 20 butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 25 wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 197 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 10 (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O. In another embodiment, Y’ is S. 5
  • R 1 is hydrogen. In another embodiment, R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen. In another embodiment, R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 10 6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen . In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a 15 nitrogen protecting group
  • Y’ is O
  • R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) 25 or phenoxyacetyl (pac)).
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 199 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- 5 butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • 10 Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl20 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S. 5
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1- 10 6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In 20 one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C 1- 6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) 25 or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 201 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S. 5
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1- 10 6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In 20 one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 202 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein each m is independently an integer selected from 1-10; Y’ is O or S, and R P and R 1 , are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6. 5
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl 15
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen 20 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O
  • R 1 is C1-6alkyl (e.g., methyl or t- 25 butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P 203 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 5 wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group 10 (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, 20 R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl 25 or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a 204 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is , 5 wherein Y’ is O or S, and R P , R 1 , L’, ZZ, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). 15 In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C1-6alkyl 25 (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a 205 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is , 5 wherein Y’ is O or S, and R P , R 1 , L’, and L are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1- 6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl). 15 In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen. In one embodiment, Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is O, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is S, R 1 is hydrogen and R P is hydrogen. In one embodiment, Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Y’ is O, R 1 is C 1- 25 6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P 206 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is , 5 wherein each m is independently an integer selected from 1-10; Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • each m is independently an integer selected from 2-10; or 2-8, or 2-6.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- 10 butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one 15 embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is O and R P is hydrogen. In one embodiment, Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). In one embodiment, Y’ is S and R P is hydrogen.
  • Boc t-butoxycarbonyl
  • Cbz benzyloxycarbonyl
  • pac phenoxyacetyl
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 20 (pac)).
  • R P is a nitrogen protecting group
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 25 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 25 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) 207 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound of Formula (X) is 5 , wherein Y’ is O or S, and R P and R 1 are as defined for Formula (X) or any embodiment thereof.
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1- 6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen.
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1- 25 6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is 208 ME1 ⁇ 53453057.v1
  • a nitrogen protecting group e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)
  • Formula (X) 5 In embodiments of Formula (X), Formula (X-a) through (X-w), including embodiments of Formulae (x-a) through (x-s) and (xi-a) through (xi-z) ZZ is a linking group formed by a reactive 209 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ZZ comprises Group(1). In some embodiments, ZZ comprises 5 Group(2). In some embodiments, ZZ comprises Group(3). In some embodiments, ZZ comprises Group(4). In some embodiments, ZZ comprises Group(5). In some embodiments, ZZ comprises Group(6).
  • ZZ comprises Group(7). In some embodiments, ZZ comprises Group(8). In some embodiments, ZZ comprises Group(9). In some embodiments, ZZ comprises Group(10). In some embodiments, ZZ comprises Group(11). In some embodiments, ZZ comprises 10 Group(12). In some embodiments, wherein ZZ is -A’-B’-A’-.
  • ZZ is -A’-B’-A’-, wherein each A’ is independently a bond, -O-, -S-, or -N(R N3 )-, wherein R N3 is independently hydrogen or C1-6alkyl and 15 each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • ZZ is CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • ZZ is C(O), C(S), or S(O)2.
  • ZZ is P(O)(OH), or P(S)(OH).
  • ZZ is –C(O)-. 20 In some embodiments, ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O-, -S-, or -N(R N3 )-; each B’ is independently CH2, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and each R N3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -A’-B’- or -B’-A’- wherein 25 each A’ is independently -O- or -N(R N3 )-, wherein R N3 is independently hydrogen or C1- 6alkyl.
  • each B’ is independently CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and 210 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO each R N3 is independently hydrogen or C1-6alkyl.
  • R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -CH2O- or -OCH2-.
  • ZZ is -S-S-.
  • ZZ is -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, - OC(O)N(R N3 )-, -N(R N3 )C(O)O-, -N(R N3 )C(O)N(R N3 )-, -S(O)2N(R N3 )-, or -N(R N3 )S(O)2-, wherein R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -C(O)N(R N3 )- or -N(R N3 )C(O)-, wherein R N3 is independently 15 hydrogen or C1-6alkyl. In some embodiments, ZZ is -C(O)O- or -OC(O)-. In some embodiments, ZZ is -OC(O)N(R N3 )-, -N(R N3 )C(O)O-, or -N(R N3 )C(O)N(R N3 )-, wherein R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -N(R N3 )C(O)N(R N3 )-, wherein R N3 is independently hydrogen 20 or C1-6alkyl. In some embodiments, ZZ is -OC(O)N(R N3 )- or -N(R N3 )C(O)O-, wherein R N3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-.
  • ZZ is -OP(O)(OH)O- or -OP(S)(OH)O-. In some embodiments, ZZ is -OP(S)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)O-. In some embodiments, ZZ is -OP(O)(OH)-, -OP(S)(OH)-, -P(O)(OH)O-, or -P(S)(OH)O-. In some embodiments, ZZ is -OP(S)(OH)- or -P(S)(OH)O-. 30 In some embodiments, ZZ is -OP(O)(OH)- or -P(O)(OH)O-.
  • the compound of Formula (X) is selected from the group consisting of: (X-a) (X-b) (X-c) (X-d) (X-e) (X-f) 212 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the compound of Formula (X) is selected from the group consisting of: 213 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 214 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 215 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 216 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the compound of Formula (X) is selected from the group consisting of: 217 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein * represents the bond to the remainder of the dsRNA agent.
  • the compound of Formula (X) is selected from the group 218 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 219 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand may be conjugated to the dsRNA agent of the disclosure, e.g., to at least one strand of the dsRNA agent of the disclosure, via a branched carrier group, e.g., a b.
  • the alpha-v-beta-6 ( ⁇ v ⁇ 6) integrin targeting ligand conjugated to the dsRNA agent of the disclosure is represented by a compound of Formula (XV): 10 ( ⁇ -ZZ-)x ⁇ -T-R T (XV) or a salt thereof, wherein x is 2, 3, 4, 5, 6, 7, or 8; T is a divalent linking group; ⁇ is a branching group; 15 each ZZ is independently -A’-B’-A’- or a linking group formed by a first reactive pair, wherein 220 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO and R 5 is -L-* wherein L is -L 1 -[G-L 2 ]q-G-L 3 -*, * is the bond to a ZZ; and q is 0 or an integer selected from 1 – 25; (e.g., 1-20, or 1-15); 5 L 1 is a bond or -B-A-; each L 2 is independently -A-B-A-; L 3 is a bond or -A-B-A-; each G is independently -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3- 10 10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; each A is independently a bond, -O-, -S-, or -N(R N )-; each B
  • embodiments for the variable ZZ are each as described above for Formula (X); embodiments for the variable R T and L are each as described above 20 for Formula (X).
  • T is a bond or **-L 6 -G 1 -[L 5 -G 1 ] q1 -L 4 -, wherein ** is the bond to R T ; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L 4 , L 5 , and L 6 are independently a bond, -A 1 -B 1 -A 1 - or ZZ 1 ; 222 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO ZZ 1 is a group formed by reaction of a reactive pair (e.g., a reaction between an azide and an alkyne or a cycloalkyne); each G 1 is independently -D 1 -E 1 -F 1 -, wherein D 1 , E 1 , and F 1 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, 5 each of which is optionally substituted with 1, 2, or 3 R groups; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-; each B 1 is independently a bond, C(O), C(S), C(NR N1 ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); each R N1 is independently hydrogen or C
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -;
  • each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond);
  • each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 15 membered heterocyclyl, aryl, or heteroaryl (e.g., C1-10alkyl or C2-10alkenyl);
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and each B 1 is
  • T is **-L 6 -G 1 -[L 5 -G 1 ]q1-L 4 -, wherein ** is the bond to R T ; q1 is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each L 4 , L 5 , and L 6 are independently a bond or -A 1 -B 1 -A 1 -, or ZZ 1 ; ZZ 1 is a group formed by reaction of an azide and alkyne or cycloalkyne; 25 each G 1 is independently C1-10alkyl, each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-; each B 1 is independently a bond, C(O), C(S), C(NR N1 ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and each R N1 is independently hydrogen or C1-6alkyl.
  • one L 5 is ZZ 1 .
  • ZZ 1 comprises: 223 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO .
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -A 1 -B 1 - or -B 1 -A 1 -; 5 each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond);
  • each G 1 is independently C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl (e.g., C 1-10 alkyl or C 2-10 alkenyl);
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alky
  • T is **-B 1 -G 1 -L 5 -G 1 -B 1 -, wherein ** is the bond to R T ; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl (e.g., C 1-10 alkyl or C 2-10 alkenyl); 15 each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alkyl; and each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -;
  • each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond);
  • each G 1 is independently C 1-10 alkyl or C 2-10 alkenyl;
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alkyl; and
  • each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -B 1 -A 1 - or -A 1 -B 1 -; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond);
  • each G 1 is independently C 1-10 alkyl or C 2-10 alkenyl;
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently 30 hydrogen or C 1-6 alkyl; and
  • each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • T is **-B 1 -G 1 -L 5 -G 1 -B 1 -, wherein ** is the bond to R T ; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C 1-10 alkyl or C 2-10 alkenyl; 224 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • T is **-B 1 -C1-10alkyl-L 5 -C1-10alkyl-B 1 -, wherein 5 ** is the bond to R T ; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -;
  • each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups;
  • 15 each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1- 6alkyl; and
  • each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ; 20 L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl; L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G 1 is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, or 3 R groups.
  • T is one of: (d) **-L 6 -G 1 -ZZ 1 -L 5 -G 1 -L 4 -, 30 (e) **-L 6 -G 1 -L 5 -G 1 -ZZ 1 -G 1 -L 4 -; or (f) **-L 6 -G 1 -ZZ 1 -G 1 -L 4 -, wherein the sum of q2 and q3 is less than q1 In certain embodiments, q2 and q3 are independently 0, 1, 2, 3 or 4.
  • each G 1 is independently C 1-20 alkyl (e.g., C 2-15 alkyl; or C 2- 12 alkyl; or C 2-10 alkyl; or C 2-8 alkyl; or C 2-6 alkyl).
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, - N(H)C(O)-, -OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is 225 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L 5 is independently -C(O)N(H)- or -N(H)C(O)-.
  • each G 1 is independently C 1-20 alkyl (e.g., C 2-15 alkyl; or C 2-12 alkyl; or C 2-10 alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - 5 OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(
  • each G 1 is independently C 1-20 alkyl (e.g., C 2-15 alkyl; or C 2-12 alkyl; or C 2-10 alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-.
  • L 6 is a bond.
  • L 6 is -C(O)-, -S(O) 2 -, - 10 P(O)(OH)-, or -P(S)(OH)-.
  • L 6 is -P(O)(OH)- or -P(S)(OH)-.
  • L 6 is -P(O)(OH)-. In certain embodiments, L 6 is -P(S)(OH)-. In certain embodiments, L 6 is -C(O)-. In certain embodiments, L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -15 OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -P(O)(OH)O-, - OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, or -N(H)C(O)-.
  • T is one of: 20 (d) **-L 6 -G 1 -ZZ 1 -L 5 -G 1 -L 4 -, (e) **-L 6 -G 1 -L 5 -G 1 -ZZ 1 -G 1 -L 4 -; or (f) **-L 6 -G 1 -ZZ 1 -G 1 -L 4 -, wherein the sum of q2 and q3 is less than q1; and ZZ 1 is selected from the group consisting of: 226 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each G 1 is independently C 1-20 alkyl (e.g., C 2-15 alkyl; or C 2-12 alkyl; or C 2-10 alkyl; or C 2-8 alkyl; or C 2- 6 alkyl).
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, - N(H)C(O)-, -OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is 10 independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L 5 is independently -C(O)N(H)- or -N(H)C(O)-.
  • each G 1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2-10alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - 15 OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(
  • each G 1 is independently C1-20 alkyl (e.g., C2-15alkyl; or C2-12alkyl; or C2-10alkyl); and each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-.
  • L 6 is a bond.
  • L 6 is -C(O)-, -S(O)2-, - P(O)(OH)-, or -P(S)(OH)-.
  • L 6 is -P(O)(OH)- or -P(S)(OH)-.
  • L 6 is -P(O)(OH)-. In certain embodiments, L 6 is -P(S)(OH)-. In certain embodiments, L 6 is -C(O)-. In certain embodiments, L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -P(O)(OH)O-, - 25 OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, or -N(H)C(O)-.
  • T is one of: (a) **-L 6 -C2-12alkyl-[L 5 -C2-12alkyl]q2-ZZ 1 -[L 5 -C2-12alkyl]q3-L 4 -; (c) **-L 6 - C2-12alkyl-L 5 -C2-12alkyl-ZZ 1 -L 5 -C2-12alkyl-L 4 -; 30 (d) **-L 6 -C 2-12 alkyl-ZZ 1 -L 5 -C 2-12 alkyl-L 4 -, (e) **-L 6 -C 2-12 alkyl-L 5 -C 2-12 alkyl-ZZ 1 -C 2-12 alkyl-L 4 -; or (f) **-L 6 -C2-12alkyl-ZZ 1 -C2-12alkyl-L 4 -, 227 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alny
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)-, - N(H)C(O)-, -OC(O)O-, -OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, 5 -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • each L 5 is independently -C(O)O-, -OC(O)-, -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, each L 5 is independently -C(O)N(H)- or -N(H)C(O)-. In certain embodiments, L 6 is a bond. In certain embodiments, L 6 is -C(O)-, -S(O) 2 -, - P(O)(OH)-, or -P(S)(OH)-. In certain embodiments, L 6 is -P(O)(OH)- or -P(S)(OH)-.
  • L 6 is -P(O)(OH)-. In certain embodiments, L 6 is -P(S)(OH)-. In certain embodiments, L 6 is -C(O)-. In certain embodiments, L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, -N(H)C(O)-, -OC(O)O-, - OC(O)N(H)-, -N(H)C(O)O-, -N(H)C(O)N(H)-, -P(O)(OH)O-, -OP(S)(OH)-, -OP(O)(OH)O, - OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -P(O)(OH)O-, - 15 OP(S)(OH)-, -OP(O)(OH)O, -OP(S)(OH)O, -P(O)(OH)O-, or -P(S)(OH)O-.
  • L 4 is -C(O)O-, -OC(O)-, -C(O)N(H)-, or -N(H)C(O)-.
  • T is selected from the following, wherein ** is the bond to R T : ( q) **-C(O)-C1-10alkyl-L5-C1-10alkylC(O)-, (r) **-C(O)-C2-10alkyl-L5-C2-10alkyl-C(O)-, 20 (s) **-C(O)-C4-10alkyl-L5-C4-10alkyl-C(O)-, (t) **-C(O)-C6-10alkyl-L5-C6-10alkyl-C(O)-, (u) **-C(O)-C2-8alkyl-L5-C2-8alkyl-C(O)-, (v) **-C(O)-C2-6alkyl-L5-C2-6alkyl-C(O)-, (w) **-C(O)-C2-4alkyl-L5-C2-4alkyl-C(O)- 25 (x
  • T is selected from the following, wherein ** is the bond to R T : 228 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO ( ff) **- N(H)C(O)-C6-12alkyl-C(O)-, (gg) **- N(H)C(O)-C10alkyl-C(O)-, (hh) **-C(O)-C2-20alkyl-C(O)N(H)-, (ii) **-C(O)-C6-20alkyl-C(O)N(H)-, 5 (jj) **-C(O)-C6-12alkyl-C(O)N(H)-, (kk) **-C(O)-C10alkyl-C(O)N(H)-, (ll) **-N(H)C(O)-C2-20alkyl-C(O)N(H)
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and 15 each G 1 is independently C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with one R group.
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and 20 each G 1 is independently C3-10cycloalkyl or 3-10 membered heterocyclyl.
  • T is -L 4 -G 1 -L 6 -**, wherein ** is the bond to R T ;
  • L 4 is -C(O)O- or C(O)N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • L 6 is -OP(O)(OH)O- or -OP(S)(OH)O-; and each G 1 is independently C3-10cycloalkyl or 3-10 membered heterocyclyl.
  • X is O or S (e.g., S).
  • T is **-L 6 -[G 5 -O]q5-G 5 -L 4 -, wherein ** is the bond to R T ;
  • q5 is an integer selected from 1 to 20;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein 30 each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl;
  • each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH);
  • T is **-C(O)-[CH2CH2-O]q5-G 5 -L 4 -, wherein ** is the bond to R T ; 35 q5 is an integer selected from 1 to 20; L 4 is -A 1 -B 1 -A 1 -, wherein 229 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is hydrogen or C1-6alkyl; each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); G 5 is C1-10alkyl.
  • T is **-C(O)-[CH2CH2-O]q5-G 5 -L 4 -, wherein ** is the bond to R T ;
  • 5 q5 is an integer selected from 1 to 20;
  • L 4 is -A 1 -B 1 or -B 1 -A 1 -, wherein each A 1 is independently -O- or -N(H)-, each B 1 is independently C(O),
  • G 5 is C1-10alkyl (e.g., C2-10alkyl or C2-6alkyl).
  • T is **-C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-, wherein ** is the bond to R T , wherein q5 is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4.)
  • Formula (XV) is according to one of Formulae (XVa) through (XVc), respectively: (XVc).
  • ⁇ is #–[G 2 -L 7 ]q2-* or #–G 3 -([L 7 -G 4 ]q3-*)y, wherein 20 # is the bond to T; y is 1, 2, 3, 4, or 5; q2 is 1, 2, 3, 4, 5, 6, 7, or 8; q3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; each G 2 , G 3 , and G 4 is independently -D 2 -E 2 -F 2 -, wherein 25 D 2 , E 2 , and F 2 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B groups, and wherein each G 2 and G 4 optionally contains at least one bond to a ZZ (e.g.,
  • each L 7 is independently -A 2 -B 2 -A 2 -; wherein each A 2 is independently a bond, -O-, -S-, or -N(R N2 )-; each B 2 is independently a bond, C(O), C(S), C(NR N2 ), S(O), S(O)2, P(O)(OH), P(S)(OH), or P(S)(SH); and 5 each R N2 is independently hydrogen, C1-6alkyl, a bond to a ZZ, or two R N2 within an -A 2 -B 2 - A 2 - group taken together with the atoms to which they are connected from a 4-8 membered heterocyclyl; and each R B is independently halogen, cyano, azido, nitro, -N(R 10 )2, -O(R 10 ), -S(R 10 ), -C(O)OR 10 , 10 -C(O)R 10 , -
  • ⁇ is #–[G2-L7]q2-* wherein # is the bond to T; q2 is 1, 2, 3, 4, 5, 6, 7, or 8; q3 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; 20 each G 2 , G 3 , and G 4 is independently -D 2 -E 2 -F 2 -, wherein D 2 , E 2 , and F 2 are independently a bond, C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B groups, and wherein each G 2 optionally contains at least one bond to ZZ; 25 each L 7 is independently -A 2 -B 2 -A 2 -; each A 2 is independently a bond, -O-, -S-, or -N(R N2 )-; each B 2 is independently a bond,
  • - ⁇ - is #–[G 2 -L 7 ]q2-*, where # is the bond to T and * is a bond to a ZZ group.
  • such embodiments include the following, wherein each * is a bond to a ZZ; # is the bond to T, each G 2 is independently C 1-10 alkyl, C 2-10 alkenyl, C 3-10 cycloalkyl, 3-10 membered 5 heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B groups; and each L 7 is independently -A 2 -B 2 -A 2 -, wherein each A 2 is independently a bond, -O-, -S-, or - N(R N2 )-; each B 2 is independently a bond, C(O), S(O) 2 , P(O)(OH), or P(S)(OH).
  • each G 2 is independently C 1-10 alkyl, each optionally substituted with 1 or 2 R B groups. In one embodiment, each G 2 is independently C 1-10 alkyl. 10 In one embodiment, each L 7 is independently -A 2 -B 2 -A 2 -, wherein each A 2 is independently a bond, -O-, -S-, or -N(R N2 )-; and each B 2 is independently a bond, C(O) or S(O) 2, provided that at least one A 2 is not a bond.
  • each L 7 is independently -A 2 -B 2 - or - B 2 -A 2 -, wherein each A 2 is independently, -O-, -S-, or -N(R N2 )-; and each B 2 is independently a bond, C(O) or S(O) 2.
  • such embodiments include each of the following, wherein # is the bond to T and each * is a bond to a ZZ group. ; and each G 2 is independently C 1-10 alkyl.
  • such embodiments include each of the following, wherein # is the bond to T and each * is a bond to a ZZ; 232 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein # is the bond to T and each * is a bond to a ZZ group.
  • # is the bond to T and each * is a bond to a ZZ group.
  • -#–G 3 -([L 7 -G 4 ] q3 -*) y where # is the bond to T and * is a bond to a ZZ group.
  • such embodiments include each of the following, 5 wherein # is the bond to T, each * is a bond to a ZZ group, and each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -C(O)O-, -OC(O)-, - C(O)N(H)-, -OC(O)O-, -N(H)C(O)O-, -OC(O)N(H)-, -OP(O)(OH)O-, or -OP(S)(OH)O-; and each G 4 is independently -D 2 -E 2 -F 2 -, wherein each D 2 and F 2 are independently a bond or C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4,
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-, -C(O)N(H)-, - OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently -D 2 -E 2 -F 2 -, wherein each D 2 and F 2 are independently a bond or C1-10alkyl; 233 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each E 2 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B groups.
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-,-C(O)N(H)-, - 5 OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently C1-10alkyl, C2-10alkenyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, 4, or 5 R B .
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, -N(H)C(O)-, -C(O)N(H)-, - 10 OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently C1-10alkyl which is optionally substituted with 1 or, 2 5 R B groups.
  • each L 7 is selected from the group consisting of -O-, -S-, -N(H)-, --N(H)C(O)-, C(O)N(H)-, - 15 OP(O)(OH)O-, and -OP(S)(OH)O-; and each G 4 is independently C1-10alkyl.
  • each L 7 is selected from the group consisting of --N(H)C(O)- and C(O)N(H)-; and each G 4 is independently C1-10alkyl.
  • such embodiments include each of the following, wherein each * is a bond to a wherein # is the bond to T and each * is a bond to a ZZ group.
  • 234 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO ZZ Embodiments, Formulae (XV) and (XVa) through (XVc)
  • ZZ is a linking group formed by a reactive pair.
  • ZZ comprises a group selected from the group consisting of 235 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • ZZ comprises Group(1).
  • ZZ comprises Group(2).
  • ZZ comprises Group(3).
  • ZZ comprises Group(4).
  • ZZ comprises Group(5).
  • ZZ comprises Group(6).
  • ZZ comprises Group(7).
  • ZZ comprises 5 Group(8).
  • ZZ comprises Group(9).
  • ZZ comprises Group(10).
  • ZZ comprises Group(11).
  • ZZ comprises Group(12). In some embodiments, wherein ZZ is -A’-B’-A’-. In some embodiments, ZZ is -A’-B’-A’-, wherein 10 each A’ is independently a bond, -O-, -S-, or -N(R N3 )-, wherein R N3 is independently hydrogen or C 1-6 alkyl and each B’ is independently CH 2 , C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH). In some embodiments, ZZ is CH 2 , C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • ZZ is C(O), C(S), or S(O) 2 . 15 In some embodiments, ZZ is P(O)(OH), or P(S)(OH). In some embodiments, ZZ is –C(O)-. In some embodiments, ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O-, -S-, or -N(R N3 )-; each B’ is independently CH 2 , C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH); and 20 each R N3 is independently hydrogen or C 1-6 alkyl.
  • ZZ is -A’-B’- or -B’-A’- wherein each A’ is independently -O- or -N(R N3 )-, wherein R N3 is independently hydrogen or C 1- 6 alkyl.
  • each B’ is independently CH 2 , C(O), S(O) 2 , P(O)(OH), or P(S)(OH); and 25 each R N3 is independently hydrogen or C 1-6 alkyl.
  • ZZ is -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, - OC(O)N(R N3 )-, -N(R N3 )C(O)O-, -N(R N3 )C(O)N(R N3 )-, -S(O)2N(R N3 )-, or -N(R N3 )S(O)2-, wherein R N3 10 is independently hydrogen or C1-6alkyl.
  • ZZ is -C(O)N(R N3 )- or -N(R N3 )C(O)-, wherein R N3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -C(O)O- or -OC(O)-. In some embodiments, ZZ is -OC(O)N(R N3 )-, -N(R N3 )C(O)O-, or -N(R N3 )C(O)N(R N3 )-, 15 wherein R N3 is independently hydrogen or C1-6alkyl.
  • ZZ is -N(R N3 )C(O)N(R N3 )-, wherein R N3 is independently hydrogen or C1-6alkyl. In some embodiments, ZZ is -OC(O)N(R N3 )- or -N(R N3 )C(O)O-, wherein R N3 is independently hydrogen or C1-6alkyl. 20 In some embodiments, ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -OP(O)(OH)-, -OP(S)(OH)-, - P(O)(OH)O-, or -P(S)(OH)O-. 25 and .
  • R T is R T1 and is as defined for Formula (X).
  • R T is -G 0 -OR T1 , wherein R T1 is as defined for Formula (X) and G 0 is selected from: 5 (c) G0 is -D0-E0-F0-, wherein D 0 and F 0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E 0 is C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; 10 (d) G0 is -D0-E0-F0-, wherein D 0 and F 0 are independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and E 0 is 3-10 membered heterocyclyl optionally substituted with 1, 2, 3, or 4 R groups; and ( e) G0 is 3-10 membered heterocyclyl optionally substituted with 1 or 2 R groups; 15 ( f)
  • G0 is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups;
  • G0 is C1-10alkyl optionally substituted with 1 or 2 R groups;
  • 5 G0 is C1-10alkyl, optionally substituted with -O(Ra), wherein Ra is independently hydrogen, C 1-6 alkyl, or a hydroxyl protecting group; e.g., , ( l) G0 is C1-10alkyl, and ( m) G0 is absent (a bond); 10 wherein * represents the bond to T, the broken bond represents the bond to OR T1 , and R is - C 1-6 alkyl-OR a or -OR a , wherein R a is independently hydrogen or C 1-6 alkyl.
  • R Y is defined in the structure of Formula (XII) according to any one of 15 the R Y Embodiments of Formula (IV).
  • Formula (XII) has the structure described above for any embodiment of Formula (IV), where the variable Z is replaced by a bond to a ZZ group.
  • Formula (XII) has the structure of any one of Formula (XII-a) through (XII-h) and (XII-t): 20 (XII-e) (XII-f) 239 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • L Embodiments Formula and (XII-a) through (XII-h) and (XII-r) In some embodiments of any one of Formula (XII) and Formulae (XII-a) through (XII-h) and 10 (XII-r), L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to a ZZ group.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, 2, 3, or 4
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, 2, or 3.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 0, 1, or 2.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, 3, 4, or 5.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, 3, or 4.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1, 2, or 3.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 1 or 2.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 4.
  • L 20 is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 3.
  • L is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein q is 2.
  • 240 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • L is -L 1 -G-L 2 -G-L 3 -*.
  • L is -L 1 -G-L 3 -*. In another embodiment, L is -G-L 3 -*. In another embodiment , L is -L 1 -G-*. In another embodiment, L is -G-*. In another embodiment, wherein L is -[G-L 2 ] q -G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q 5 is 2; or q is 3; or q is 4; or q is 5).
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)O-, -OC(O)N(R N )-, - N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -OP(O)(OH)O- ,-OP(S)(OH)O-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-,- N(R N )C(O)N(R N )-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)N(R N )-, -N(R N )C(O)-, -OC(O)N(R N )-, -N(R N )C(O)O-, -N(R N )C(O)N(R N )-, -O-, 15 and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each instance of A-B-A- is independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • each instance of A-B-A- is independently selected from the group 20 consisting of -C(O)N(R N )-, -N(R N )C(O)-, -O-, and -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl.
  • D and F are each independently a bond, C1-10alkyl, C2-10alkenyl, or C2-10alkynyl, each optionally substituted with 1, 2, 3, or 4 R groups; and 25 E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • D and F are each independently a bond or C1-10alkyl optionally substituted with 1, 2, 3, or 4 R groups; and 30 E is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3- 10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • each G is independently C1-10alkyl, optionally substituted with 1, 2, or 3 R groups. In some embodiments, each G is independently C1-10alkyl, optionally substituted with 1 or 2 R groups. 241 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each G is independently C1-10alkyl, optionally substituted with one R group
  • L is -L 1 -G-L 3 -*, wherein * is the bond to a ZZ group ;
  • G is -D-E-F-, wherein D, E, and F are independently a bond, C1-10alkyl, C2-10alkenyl, C2- 5 10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups;
  • L 1 is -B-A-;
  • L 3 is a bond or -A-B-A-;
  • each A is independently a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently 10 hydrogen or C1-6alkyl.
  • each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH); and
  • L is -L 1 -G-L 3 -*, wherein * is the bond to a ZZ group; G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1, 2, 3, or 4 R groups; 15 L 1 is -B-; L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-, wherein each R N is independently hydrogen or C1-6alkyl; and each B is independently a bond, CH2, C(O), S(O)2, P(O)(OH), or P(S)(OH; and 20 In some embodiments, L is -L 1
  • L is -L 1 -G-*, wherein * is the bond to a ZZ group; G is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1 or 2 R groups; 30 L 1 is -B-A-, wherein A is a bond, -O-, -S-, or -N(R N )-, each R N is independently hydrogen or C1-6alkyl; and B is a bond, C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • L is -L 1 -G-*, wherein * is the bond to a ZZ group; G is C1-10alkyl or C2-10alkenyl, each of which is optionally substituted with 1 or 2 R groups; 35 L 1 is bond, C(O), S(O)2, P(O)(OH), or P(S)(OH); and R N is hydrogen or C1-6alkyl.
  • L is wherein * is the bond to a ZZ group; k is an integer from 1 to 10; L 1 is bond, C(O), C(S), C(NR N ), S(O) 2 , P(O)(OH), or P(S)(OH); and R N is hydrogen or C 1-6 alkyl.
  • L is wherein * is the bond to a ZZ group; k is an 5 integer from 1 to 10; L 1 is bond, C(O), P(O)(OH), or P(S)(OH).
  • L is , wherein * is the bond to a ZZ group; k is an integer from 1 to 10; or an integer from 2 to 10; or an integer from 3 to 10; or an integer from 4 to 10; or an integer from 5 to 10; or an integer from 5 to 9; or an integer from 5 to 8; or an integer from 5 to 7. 10 In some embodiments, L is , wherein * is the bond to a ZZ group; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3).
  • t is an integer from 0 to 10 (e.g., an integer from 1 to 5 or 1; or 2; or 3); a is an integer from 1 to 3; and s and s’ are each independently an integer from 1 to 15 24 (e.g., an integer from 1 to 16; an integer from 1 to 10; an integer from 3 to 10; an integer from 3 to 7; or an integer from 4 to 6).
  • * is the bond to a ZZ group; a is 1, 2 or 3; and each s, s’, and s” independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer20 from 4 to 6).
  • L is wherein * is the bond to a ZZ group; and s, s’, and s’’ are independently is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • each s, s’, and s independently is an integer from 1 to 24(e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7, or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ; s and 5 k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is wherein * is the bond to ZZ 10 and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6).
  • L is C1-20 alkyl. In some embodiments, L is C2-20 alkyl. In some embodiments, L is C6-20 alkyl. In some embodiments, L is C8-12 alkyl. In some embodiments, L is C10 alkyl. 15 In another embodiment, wherein L is -[G-L 2 ]q-G-*, wherein q is 1, 2, 3, 4, or 5 (e.g., q is 2; or q is 3; or q is 4; or q is 5), each G is indepdently C 1-10 alkyl and L 2 is O, S, or N(H).
  • each G is indepdently C1-10alkyl and L 2 is O.
  • L is -[CH 2 CH 2 O] q -G-*, wherein q is 1, 2, 3, 4, or 520 (e.g., q is 2; or q is 3; or q is 4; or q is 5)
  • G is indepdently C 1-10 alkyl.
  • L is , wherein * is the bond to ZZ; t is an integer from 0 to 10 (e.g., an integer from 1 to 5; or 1; or 2; or 3). I n some embodiments, L is , wherein * is the bond to ZZ. In some embodiments, L is , wherein * is the bond to ZZ; t is 1 to 10 25 (e.g., 1-5; or 1-3; or 1; or 2; or 3); and z is 1-10 (e.g., 1-6; or 1-4; or 1; or 2; or 3 or 4 or 5 or 6). 244 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • L is , wherein * is the bond to ZZ, and t is an integer from 1 to 5 (e.g., 1; or 2; or 3). I n some embodiments, , wherein * is the bond to ZZ.
  • R L Embodiments, Formulae (XII) and (XII-a) through (XII-h) and (XII-r) 5 In some embodiments of any one of Formula (XII) and Formulae (XII-a) through (XII-h) and (XII-r), R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen or C1-6alkyl and R 4 is R 5 .
  • R L is -N(R 3 )(R 4 ), wherein R 3 is hydrogen and R 4 is R 5 . In some embodiments, R L is -N(R 3 )(R 4 ), wherein R 3 is C1-3alkyl and R 4 is R 5 . In some embodiments, R L is -N(R 3 )(R 4 ), wherein R 3 is methyl and R 4 is R 5 . 10 In some embodiments, R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that is substituted with R 5 .
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a 4 – 8 membered monocyclic heterocyclyl group that 15 is substituted with R 5 , provided that R 3 and R 4 taken together with the nitrogen atom to which they are attached do not form a morpholino group.
  • R L is --N(R 3 )(R 4 ), wherein R 3 and R 4 taken together with the nitrogen atom to which they are attached form a group that is piperidinyl, piperazinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, azetidinyl, pyrrolinyl, imidazolinyl, or 20 pyrazolinyl, each substituted wi In some embodiments, .
  • R L is 25 2, 3, 4, 5, 6, 7, 8, 9 or 10; a is an integer from 1 to 3 and s and s’ are each independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6). 245 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • R L is 10 wherein * is a bond to a ZZ group; s, s’, and s’’ are independently an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and t is an integer from 1 to 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, an integer from 1 to 3, or 1, or 2, or 3).
  • R L is , wherein * is a bond to a ZZ group; s is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 t 20 , wherein * is a bond to a ZZ group; s is an integer from 1 to 24 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and t is an integer from 0 to 10 (e.g., an integer from 1 to 8, an integer from 1 to 5, an integer from 1 to 3, or 1, or 2, or 3). 25 In some embodiments, R L is -R 5 .
  • RL is , wherein * is a bond to a ZZ group; t 5 is 0 to 10 (e.g., 1-5; or 1-3; or 1 I n some embodiments, wherein * is a bond to a ZZ group; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to10 10, an integer from 3 to 7 or an integer from 4 to 6).
  • R L is wherein * is a bond to a ZZ group.
  • R L is 247 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein * is a bond to a ZZ group and w is an integer selected from 1-10 (e.g., 2-10, 2-8, 4-8). . 5
  • the compound of Formula (XII) is according to one of Formulae (XII-i) through (XII-j) 248 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each R 21 is independently selected from group consisting of R and a nitrogen protecting group, 5 and L is defined according to Formula (XII), or according to any of the preceding embodiments of L.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • the compound of Formula (XII) is according to one of Formulae (XII- m) through (XII-q) and (XII-t): 249 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO or a salt thereof, wherein * is a bond to a ZZ group, p is 0, 1 or 2; each R 21 is independently selected from group consisting of R; R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), 5 and L is defined according to Formula (XII), or according to any of the preceding embodiments of L, wherein R and the remaining variables are as defined in Formula (XII).
  • the compound of Formula (XII) is according to one of Formulae (XII-u) through (XII-z): (XII-u) (XII-v) 250 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • R 21 is independently selected from group consisting of R;
  • R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group), and L is defined according to Formula (XII), or according to any of the preceding 5 embodiments of L, wheren R and the remaining variables are as defined in Formula (XII).
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • R N3 is independently hydrogen or C 1-6 alkyl; ⁇ is selected from, 251 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein # is the bond to T and each * is a bond to a ZZ group; and wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through (XII-z);
  • ZZ is - C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C 1-6 alkyl;
  • 10 ⁇ is selected from, wherein # is the bond to T and each * is a bond to a ZZ group. 15 and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • R N3 is independently hydrogen or C1-6alkyl
  • ⁇ is selected from, ⁇ wherein # is the bond to T and each * is a bond to a ZZ group, G 3 is C1-10alkyl; 10 each G 4 is independently C1-10alkyl; and , wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • 15 ⁇ is selected from Formula (XII) and any one of Formula (XII-a) through (XII-q) and (XII-t) through (XII-z); ZZ is -C(O)-, -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C1-6alkyl; ⁇ is selected from, , , , 253 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-i) through (XII-q) and (XII-t) through (XII-z);
  • ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C1-6alkyl;
  • 10 T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -B 1 -A 1 - or -A 1 -B 1 -; 254 ME1 ⁇ 53453057.v1
  • each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C1-10alkyl or C2-10alkenyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and 5 each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH); and wherein ** represents the bond to T, and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • 10 ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z);
  • ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, wherein R N3 is independently hydrogen or C1-6alkyl;
  • 15 T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ;
  • L 4 and L 6 are independently -B 1 -A 1 - or -A 1 -B 1 -; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - (e.
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z); ZZ is -C(O)N(H)- or -N(H)C(O)-; 5 ⁇ is T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ; L 4 is -A 1 -B 1 -; L 6 is -B 1 -; L 5 is a bond, -B 1 -A 1 -, or -A 1 -B 1 -; 10 each G 1 is independently C1-10alkyl; each A 1 is independently a bond, -O- or -N(H); bond t ,
  • R P3 is hydrogen; 256 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII- t); ZZ is -C(O)N(H)- or -N(H)C(O)-; and T is **-L 6 -G 1 -L 5 -G 1 -L 4 -, wherein ** is the bond to R T ; L 4 is -A 1 -B 1 -; 5 L 6 is -B 1 -; L 5 is a bond, -B 1 -A 1 -, or -A 1 -B 1 -; each G 1 is independently C1-10alkyl; each A 1 is independently a bond, -O- or -N(H); each
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) through (XII-z);
  • ZZ is -OP(O)(OH)O-, -OP(S)(OH)O-, -C(O)N(R N3 )-, -N(R N3 )C(O)-, -C(O)O-, -OC(O)-, 15 wherein R N3 is independently hydrogen or C1-6alkyl;
  • T is ***-L 6 -[G 5 -O] q5 -G 5 -L 4 -, wherein q5 is an integer selected from 1 to 20;
  • L 4 and L 6 are independently -A 1 -B 1 -A 1 -, wherein 20 each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) 5 through (XII-z), wherein L is wherein * is the bond to ZZ; s and k are independently is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); and w is an integer from 1 to 10 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6); ; 10 ZZ is -C(O)N(H)-, -N(H)C(O)-; T is ***-L 6
  • ⁇ is selected from Formula (XII) and any one of Formula (XII-m) through (XII-q) and (XII-t) t hrough (XII-z), wherein L is wherein * is the bond to ZZ and w is an integer from 1 to 20 (e.g., an integer from 1 to 16, an integer from 1 to 10, an integer from 3 to 10, an integer from 3 to 7 or an integer from 4 to 6; 258 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO ZZ is -C(O)N(H)-, -N(H)C(O)-; T is **-C(O)-[CH2CH2-O]q5- C2-10alkyl-C(O)N(H)-, wherein ** is the bond to the pyrrolidine ring and q5 is an integer selected from 1 to 20 (e.g., 1 to 10, or 2 to 10; or 2 – 8; or 1; or 2; or 3; or 4); and the remaining variables are as defined in Formula (XV), and any embodiment thereof.
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 3’-end of the 10 oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on a internal nucleotide (i.e., a nucleotide that is not the 5’-terminal or 3’-terminal nucleoside).
  • L L is a divalent linker that connects to the 3’-end of the 10 oligonucleotide, the 5’-end of the oligonucleotide, or an internal 2’- or 3’ position on a internal nucleotide (i.e., a nucleotide that is not the 5’-terminal or 3’-terminal nucleoside).
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; 15 directly to the 3’-O of the 3’-terminal nucleoside; directly to the 4’-carbon of the 3’-terminal nucleoside; directly to the 2’-carbon of the 3’-terminal nucleoside; or directly to the 2’-O of the 3’-terminal nucleoside.
  • L L is a divalent linker that connects to the 3’-end of the oligonucleotide, such as one of : directly to the 3’-carbon of the 3’-terminal nucleoside; 15 directly to the 3’-O of the 3’-terminal nucleoside; directly to the 4’-carbon of the 3’-terminal nucleoside; directly to the 2’-carbon
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that 20 connects to the 5'-end of the oligonucleotide, such as one of: directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of the 5’-terminal nucleoside; or 25 directly to the 2’-O of the 5’-terminal nucleoside.
  • L L is a divalent linker that 20 connects to the 5'-end of the oligonucleotide, such as one of: directly to the 5’-carbon of the 5’-terminal nucleoside; directly to the 5’-O of the 5’-terminal nucleoside; directly to the 4’-carbon of the 5’-terminal nucleoside; directly to the 2’-carbon of
  • R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internal 2’- or 3’ position on an internal nucleotide. In some embodiments, R T1 is -L L -oligonucleotide, wherein L L is a divalent linker that connects to an internal 2’- position on a internal nucleotide.
  • L L when L L connects to a carbon aton on a nucleoside, then L L is -B 3 -A 3 -, wherein B 3 is -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH)-; and A 3 is -O-, -S-, or -N(H)- .
  • L L when L L connects to a carbon atom on a nucleoside, then L L is -B 3 - 35 A 3 -, wherein B 3 is -P(O)(OH)- or-P(S)(OH)-; and A 3 is -O-.
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH). In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)- or -P(S)(OH)-. 5 In another embodiment, when L L connects to a oxygen atom on a nucleoside, then L L is - P(O)(OH)-.
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is - P(S)(OH)-. In another embodiment, when L L connects to a oxygen atom on a nucleoside, then 10 LL is -B3-A3-LL1-A3-B3-, wherein B 3 is a bond, -C(O)-, C(S)-, C(NH), S(O), S(O)2, -P(O)(OH)-, -P(S)(OH)-, or -P(S)(SH); A 3 is a bond, -O-, -S-, or -N(H)- ; and L L1 is C1-10alkyl, C2-10alkenyl, C2-10alkynyl, C3-10cycloalkyl, 3-10 membered heterocyclyl, aryl, or heteroaryl.
  • LL when LL connects to a oxygen atom on a nucleoside, then LL is -B3- L L1 -B 3 -, wherein B 3 is -C(O)-; and L L1 is C1-10alkyl.
  • R T is R T1
  • R T1 is -L L -oligonucleotide
  • the nucleoside is of Formula (XV-f), (XV-f) wherein B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- 20 methylcytosine, or-5-methyluracil); Tis according any of the preceding embodiments; and * represent the bond to ZZ.
  • -T- is -L 1 -G-L 3 -*, wherein * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one or two R groups, and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein the alkyl 25 is optionally substituted with one or two groups selected from the group consisting of halogen, hydroxy, C1-6alkoxy, amino, Cl-6alkylamino, di(C1-6alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with one group selected from the group consisting of halogen, hydroxy, C1-6alkoxy, amino, Cl-6alkylamino, di(C1- 6alkylamino), cyano, carboxy , and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, 30 wherein the alkyl is optionally substituted with one group selected from the group consisting of hydroxy, amino, and carboxy , and * is the bond to ZZ.
  • -T- is -C2-30alkyl-*, wherein the alkyl is optionally substituted with hydroxy, and * is the bond to ZZ.
  • 260 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • -T- is -C2-30alkyl-*, wherein * is the bond to ZZ.
  • -T- is -C2-16alkyl-*, wherein * is the bond to ZZ.
  • -T- is -C4-12alkyl-*, wherein * is the bond to ZZ.
  • -T- is -C4-10alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C5-10alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C6alkyl- 5 *, wherein * is the bond to ZZ. In some embodiments, -T- is -C8alkyl-*, wherein * is the bond to ZZ. In some embodiments, -T- is -C10alkyl-*, wherein * is the bond to ZZ.
  • -T- is -L 1 -[G-L 2 ]q-G-L 3 -*, wherein * is the bond to ZZ; q is 0, 1, 2, 3, 4, or 5; 10 L 1 is a bond or -B-A-; each L 2 is independently -A-B-A-; L 3 is a bond or -A-B-A-; each A is independently a bond, -O-, -S-, or -N(R N )-; each B is independently a bond, CH2, C(O), C(S), C(NR N ), S(O)2, P(O)(OH), or P(S)(OH); 15 each R N is independently hydrogen or C1-6alkyl; and each G is independently C1-10alkyl, C2-10alkenyl, C2-10alkynyl, each of which is optionally substituted with 1, 2, 3, or 4 R groups.
  • -T- is -L 1 -[G-L 2 ]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3;
  • -T- is -[G-L 2 ]q-G-*, wherein * is the bond to ZZ, q is 0, 1, 2, or 3 (e.g., q is 0, 1, or 2; or 0 or 1; or 0; or 1; or 2); each G is independently C1-10alkyl, each of which is optionally 30 substituted with 1 or 2 R groups.
  • each L2 is independently C(O)O or OC(O);
  • each L2 is independently C(O)(NRN) or N(RN)C(O), wherein each RN is independently hydrogen or C1-6alkyl
  • each L2 is independently OP(O)(OH)O, or OP(S)(OH)O (e.g., each is OP(O)(OH)O); 35 or ( f) each L2 is a bond.
  • the compound of Formula (XV) is according to one of Formulae (XV- g) through (XV-q): 261 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil); each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); 5 each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6). 262 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • nucleobase e.g., adenine, cytosine, uracil, guanine, 5- methylcytosine, or-5-methyluracil
  • each n is independently 0 or an integer selected from 1-10; (e.g., 1-5, or 1-3, or 3, or 2, or 1); 5 each m is
  • the compound of Formula (XV) is according to one of Formulae (XV- r) through (XV-w): 263 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO or a salt thereof wherein L and ZZ are as defined in Formula (XV) or in any embodiment preceding or below;
  • B is an optionally modified nucleobase (e.g., adenine, cytosine, uracil, guanine, 5- 5 methylcytosine, or-5-methyluracil); each m is independently integer selected from 1-20 (e.g., 2-12, or 2-10; or 2-6; or 2; or 3; or 4; or 5; or 6); 264 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO R P is hydrogen or a nitrogen protecting group (e.g., a nitrogen protecting group); and R 1 is hydrogen or C1-6alkyl (e.g., methyl or t-butyl).
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • R 1 is hydrogen and R P is hydrogen.
  • R 1 is hydrogen and a nitrogen protecting group.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl) and a nitrogen protecting group.
  • R T is R T1 , wherein R T1 is -L L -oligonucleotide
  • the internucleotide linkage can be of the formula, including the 3’ and 5’ oxygen atoms of the preceding and following nucelosides, respectively, (XV-pa) (XV-pb) (XV-pc) wherein T can be, for example a bond, -S(O)2- or, in for Formula (XV-i), a 5 -8 membered 15 heterocyclyl ring optionally substituted with 1 or 2 R groups, as defined herein ; and * represent the bond to ⁇ .
  • the preceding includes, (XV-pd) (XV-pe) (XV-pf) wherein * represent the bond to ⁇ ; and R N5 is hydrogen or C1-10 alkyl.
  • the preceding includes, (XV-pg) (XV-ph) (XV-pi) 20 wherein * represent the bond to ⁇ ; m is an integer selected from 1 – 20 (e.g., 1-10, or 2-20, or 2-10, or 4-10, or 4-8; or 6-12; or 5; or 6; or 7; or 8; or 9; or 10), and R N5 is hydrogen or C1-10 alkyl.
  • the compound of Formula (XV-pd) is 265 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R Y , Y, R 1 , L, T, ⁇ , and ZZ are as defined for Formula (XV).
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl). 5 266 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO wherein Y’ is O or S, and R 1 , T, ⁇ , R P , L, and ZZ are as defined for Formula (XV).
  • the compound is 267 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), Y’ is O or S, and R 1 , ⁇ , R P and ZZ are as defined for Formula (XV).
  • the compound of Formula (XV-pe) is 268 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R Y , Y, R 1 , L, T , ⁇ , and ZZ are as defined for Formula (XV).
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- 5 butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C 1- 6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • 10 Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 15 (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R P is a nitrogen protecting group
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 20 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C 1-6 alkyl (e.g., methyl or t-butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C 1-6 alkyl (e.g., methyl or t-butyl)
  • R P is a 25 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound is 269 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 270 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO 271 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein Y’ is O or S, and R 1 , L, T, ⁇ , R P , and ZZ are as defined for Formula (XV).
  • Y’ is O.
  • Y’ is S. 5
  • R 1 is hydrogen.
  • R 1 is C 1-6 alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C 1- 10 6 alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C 1-6 alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a 15 nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is hydrogen
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) 5 and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound is 10 273 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO 5 wherein each m is an integer selected from 1 – 20 (e.g., 2-20, 2-10, 1-10, 2-16, 4-16, 4-8, or 6-12), Y’ is O or S, and R 1 , ⁇ , R P , and ZZ are as defined for Formula (XV).
  • Y’ is O.
  • Y’ is S. 274 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t- butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O and R 1 is hydrogen.
  • R 1 is C1- 6alkyl (e.g., methyl or t-butyl).
  • Y’ is S and R 1 is hydrogen.
  • Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl 10 (pac)).
  • Y’ is S and R P is hydrogen .
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen 15 protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen .
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t-butyl) 20 and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • L L when L L connects to a oxygen atom on a nucleoside, then L L is-a 25 bond.
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 5’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-5’) (XV-5’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to 30 remainder of the compound of Formula (XV).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 5’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-5’o) or Formula ( 35 wherein * represents the bond to remainder of the compound of Formula (XV).
  • a compound of Formula (XV) can be represented by, 275 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated at the 3’-end of the 5 oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-3’) wherein L L is -P(Y)(OH)-, wherein Y is O or S (e.g., S); and * represents the bond to remainder of the compound of Formula (XV).
  • R T1 when R T1 is -L L -oligonucleotide and is conjugated 10 at the 3’-end of the oligonucleotide, in one embodiment, R T1 can be represented as Formula (XV-3’o) or Formula ( wherein * represents the bond to remainder of the compound of Formula (XV).
  • a compound of Formula (XV) can be represented by, 15 , wherein Y’ is O or S, x is an integer selected from 2 to 8, and ⁇ , R Y , Y, R 1 , L, T, and ZZ are as defined for Formula (XV) or any embodiment thereof.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • the compound of Formula (XV) is 276 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO 5 wherein m is an integer selected from 1 – 10; n in an integer selected from 1 – 6; L 4 and L 6 are independently -B 1 -A 1 - or -A 1 -B 1 -; each L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C1-10alkyl or C2-10alkenyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently 10 hydrogen or C1-6alkyl; and each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • each ⁇ is , wherein m is an integer selected from 1 – 10. 277 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • each ⁇ is , wherein m is an integer selected from 1 – 10;
  • each 5 wherein m is an integer selected from 1 – 10;
  • each , wherein m is an integer selected from 1 – 10.
  • each ⁇ is 10 , wherein m is an integer selected from 1 – 10; 278 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein m is an integer selected from 1 – 10; and n is an integer selected from 1 – 6. 5 wherein m is an integer selected from 1 – 10; and n is an integer selected from 1 – 6. I n another embodiment of Formula (XV-x), each . In another embodiment of Formula (XV-x), each .
  • L 4 and L 6 are independently -B 1 -A 1 - or -A 1 -B 1 -; 279 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO L 5 is a bond, -B 1 -A 1 - or -A 1 -B 1 -; each G 1 is independently C1-10alkyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and 5 each B 1 is independently a bond, C(O), C(S), S(O)2, P(O)(OH), or P(S)(OH).
  • L 4 is -B 1 -A 1 - or -A 1 -B 1 -;
  • L 6 is B 1 ;
  • L 5 is a bond, -B 1 -A 1 - or -A 1 -B 1 -;
  • each G 1 is independently C1-10alkyl;
  • each A 1 is independently -O- or -N(H)-, and
  • each B 1 is independently C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • L 5 is a bond, -B 1 -A 1 - or -A 1 -B 1 -; each A 1 is independently -O- or -N(H)-, and each B 1 is independently C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • -L 4 -G 1 -L 5 -G 1 -L 4 - is -C(O)-C1-10alkyl-L 5 -C1-10alkyl-C(O)N(H)-, wherein L 5 is a bond, -B 1 -A 1 - 20 or -A 1 -B 1 -, wherein A 1 is -O- or -N(H)-, and B 1 is C(O), S(O)2, P(O)(OH), or P(S)(OH).
  • the compound of Formula (XV) is 25 wherein Y’ is O or S; each ZZ is N(H)C(O) or C(O)N(H); 280 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • each G 1 is independently C1-10alkyl; 5 each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C1-6alkyl; and wherein m is an integer selected from 1 – 10 R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is 10 hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • each ⁇ is , wherein m is an integer selected from 1 – 10 ; R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., 15 hydrogen). ; 281 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • the compound of Formula (XV) is wherein Y’ is O or S; e 10 wherein m is an integer selected from 1 – 10; n is an integer selected from 1-6; L 5 is a bond or - A 1 -B 1 -A 1 - (e.g., a bond, -B 1 -A 1 - or -A 1 -B 1 -; or a bond); each G 1 is independently C 1-10 alkyl; each A 1 is independently a bond, -O-, -S-, or -N(R N1 )-, wherein R N1 is independently hydrogen or C 1-6 alkyl; and 15 each B 1 is independently a bond, C(O), C(S), S(O) 2 , P(O)(OH), or P(S)(OH).
  • each ⁇ is hydrogen).
  • each ⁇ is , wherein m is an integer selected from 1 – 10; ; R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • each ⁇ is , wherein m is an integer selected from 1 – 10; R 1 is hydrogen or C1-6alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g.,15 hydrogen). 285 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • each ⁇ is , wherein m is an integer selected from 1 – 10 R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • each ⁇ is 5 hydrogen).
  • each , wherein m is an integer selected from 1 – 10; and n is an integer selected from 1 – 6; R 1 is hydrogen or10 C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen).
  • each ⁇ is 10 , wherein R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen) .
  • R 1 is hydrogen or C 1-6 alkyl (e.g., hydrogen); and R P is hydrogen or a nitrogen protecting group (e.g., hydrogen) .
  • Y’ is O.
  • Y’ is S.
  • R 1 is hydrogen.
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl).
  • R P is hydrogen.
  • R P is a nitrogen protecting group (e.g., t- butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)). 287 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • Y’ is O and R 1 is hydrogen.
  • Y’ is O and R 1 is C1-6alkyl (e.g., methyl or t-butyl). In one embodiment, Y’ is S and R 1 is hydrogen. In one embodiment, Y’ is S and R 1 is C1-6alkyl (e.g., methyl or t-butyl). 5 In one embodiment of Formula (XV-x), (XV-x1), and (XV-x2), and each of the preceding embodiments thereof, Y’ is O and R P is hydrogen .
  • Y’ is O and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S and R P is hydrogen.
  • Y’ is S and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • R 1 is hydrogen and R P is hydrogen.
  • Y’ is S, R 1 is hydrogen and R P is hydrogen.
  • Y’ is O, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S, R 1 is hydrogen and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl 15 (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is O, R 1 is C1- 6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is S, R 1 is C1-6alkyl (e.g., methyl or t-butyl) and R P is hydrogen.
  • Y’ is O, R 1 is C1-6alkyl (e.g., methyl or t- butyl) and R P is a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • Y’ is S
  • R 1 is C1-6alkyl (e.g., methyl or t-butyl)
  • R P is 20 a nitrogen protecting group (e.g., t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) or phenoxyacetyl (pac)).
  • the compound is of the structure: wherein each 25 1-10; 2-10; 2-6; 4; 3; 2; or 1) ; and 288 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • the compound is of the structure: 5 10
  • the compound is of the structure: 289 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO 5
  • the compound is of the structure: 290 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO , wherein * represents the bond to the remainder of the dsRNA agent.
  • the compound of Formula (XV) is 5 291 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein * represents the bond to the remainder of the dsRNA agent.
  • the compound of Formula (XV) is, 5 wherein * represents the bond to the remainder of the dsRNA agent.
  • the compound of Formula (XV) is, 10 292 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO wherein * represents the bond to the remainder of the dsRNA agent.
  • the compound of Formula (XV) is, 5 wherein * represents the bond to the remainder of the dsRNA agent. 293 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • the compound of Formula (XV) is, wherein B is an optionally modified nucleobase (e.g., cytosine, uracil, 5-methylcytosine, 5- 5 wherein * represents the bond to the remainder of the dsRNA agent. 10 VI.
  • RNAi agent of the disclosure Delivery of an RNAi Agent of the Disclosure
  • a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a target gene-associated (i.e., DMPK-associated) disorder
  • delivery 15 may be performed by contacting a cell with an RNAi agent of the disclosure either in vitro or in vivo.
  • In vivo delivery may also be performed directly by administering a composition comprising an RNAi agent, e.g., a dsRNA, to a subject.
  • in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent.
  • any method of delivering a nucleic acid molecule in vitro or in vivo can be adapted for use with a RNAi agent of the disclosure.
  • factors to consider in order to deliver an RNAi agent include, for example, biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue.
  • the RNA can be modified or alternatively 25 delivered using a drug delivery system; both methods act to prevent the rapid degradation of the 294 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO dsRNA by endo- and exo-nucleases in vivo.
  • RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.
  • the RNAi agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system.
  • a RNAi agent forms a complex with cyclodextrin for systemic administration.
  • Certain aspects of the instant disclosure relate to a method of reducing the expression of a target gene, i.e., DMPK, in a cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure.
  • the cell is a skeletal muscle.
  • the cell is a cardiac muscle cell.
  • the cell is an adipocyte.
  • the RNAi agent is taken up on one or more tissue or cell types present in organs, e.g., liver, skeletal muscle, cardiac muscle, adipose tissue.
  • Another aspect of the disclosure relates to a method of reducing the expression and/or activity of a target gene in a subject, comprising administering to the subject the double-stranded RNAi agent of the disclosure.
  • Another aspect of the disclosure relates to a method of treating a subject having a target gene- associated disorder or at risk of having or at risk of developing a target gene-associated disorder, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the disclosure, thereby treating the subject.
  • the double-stranded RNAi agent is administered subcutaneously. 25 In one embodiment, the double-stranded RNAi agent is administered intramuscularly. In one embodiment, the double-stranded RNAi agent is administered by intravenously.
  • modified siRNA compounds e.g., modified siRNA compounds
  • a composition that includes a RNAi agent can be delivered to a subject by a variety of routes.
  • Exemplary routes include pulmonary system, intravenous, subcutaneous, oral, topical, rectal, anal, vaginal, nasal, and ocular.
  • the RNAi agents of the disclosure can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically include one or more species of RNAi agent 35 and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active 295 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • compositions of the present disclosure may be administered in a number 5 of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be intratracheal, intranasal, topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, parenteral, or pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer.
  • Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection administration. 10
  • the route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice.
  • Lung cells might be targeted by administering the RNAi agent in powder or aerosol form.
  • the vascular endothelial cells could be targeted by coating a balloon catheter with the RNAi agent and mechanically introducing the RNA.
  • Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorption enhancers and other suitable additives. Such administration permits both systemic and local delivery of the double stranded RNAi agents of the disclosure.
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • Conventional pharmaceutical 20 carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves, and the like may also be useful.
  • Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
  • carriers that can be used include lactose, sodium citrate and salts of phosphoric acid.
  • the administration of the siRNA compound is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), 35 intradermal, intraperitoneal, intramuscular, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary system, intranasal, urethral, or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the 296 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No.
  • ALN-529-WO medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
  • Vector encoded RNAi agents of the Disclosure 5 RNAi agents targeting the target gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114, and US 6,054,299). Expression can be sustained (months or longer), depending upon the specific construct used and the target tissue or cell type.
  • transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating 10 vector.
  • the transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).
  • the individual strand or strands of a RNAi agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target 15 cell.
  • each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid.
  • a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
  • RNAi agent expression vectors are generally DNA plasmids or viral vectors. Expression 20 vectors compatible with eukaryotic cells, such as those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a RNAi agent as described herein.
  • RNAi agent expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
  • Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno- associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors 30 or avipox, e.g.
  • RNAi agent canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication- defective viruses can also be advantageous.
  • Different vectors will or will not become incorporated into the cells’ genome.
  • the constructs can include viral sequences for transfection, if desired.
  • the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors.
  • Constructs for the recombinant expression of a RNAi agent will generally require 35 regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the RNAi agent in target cells.
  • Other aspects to consider for vectors and constructs are known in the art. VII.
  • compositions 297 ME1 ⁇ 53453057.v1 Attorney Docket No.121301-24020 Alnylam Reference No. ALN-529-WO
  • the present disclosure also includes pharmaceutical compositions and formulations which include the RNAi agents of the disclosure.
  • pharmaceutical compositions containing an RNAi agent, as described herein, and a pharmaceutically acceptable carrier are useful for treating a subject 5 who would benefit from inhibiting or reducing the expression of a target gene, e.g., a subject having a DMPK–associated disorder, e.g., DM1.
  • Such pharmaceutical compositions are formulated based on the mode of delivery.
  • compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery.
  • IV intravenous
  • IM intramuscular
  • subQ subcutaneous
  • the pharmaceutical compositions of the disclosure are pyrogen free or non-pyrogenic.
  • the delivery vehicle can deliver an iRNA compound, e.g., a double- stranded iRNA compound, or ssiRNA compound, (e.g., a precursor thereof, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA 15 compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) to a cell by a topical route of administration.
  • the delivery vehicle can be microscopic vesicles.
  • the microscopic vesicles are liposomes.
  • the liposomes are cationic liposomes.
  • the microscopic vesicles are micelles.
  • the disclosure features a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA 20 compound, or ssiRNA compound, (e.g., a precursor thereof, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) in an injectable dosage form.
  • the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders.
  • the sterile 25 solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.
  • a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor thereof, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA 30 which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) in oral dosage form.
  • siRNA compound e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof
  • a pharmaceutical composition including an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor thereof, e.g.
  • the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules.
  • the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach.
  • the enteric material 35 is a coating.
  • the coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate.
  • the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

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Abstract

L'invention concerne des agents d'acide ribonucléique double brin (ARNdb) et des compositions ciblant un gène de protéine kinase myotonique de dystrophie (DMPK), ainsi que des procédés d'inhibition de l'expression d'un gène DMPK et des méthodes de traitement de sujets ayant une maladie ou un trouble associé à DMPK, par exemple, la dystrophie myotonique (DM), à l'aide de tels agents et compositions d'ARNdb.
PCT/US2025/033136 2024-06-12 2025-06-11 Compositions d'arni de protéine kinase myotonique de dystrophie (dmpk) et leurs procédés d'utilisation Pending WO2025259747A2 (fr)

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Citations (157)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4845205A (en) 1985-01-08 1989-07-04 Institut Pasteur 2,N6 -disubstituted and 2,N6 -trisubstituted adenosine-3'-phosphoramidites
US4981957A (en) 1984-07-19 1991-01-01 Centre National De La Recherche Scientifique Oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US5130300A (en) 1986-03-07 1992-07-14 Monsanto Company Method for enhancing growth of mammary parenchyma
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5177195A (en) 1991-01-08 1993-01-05 Imperial Chemical Industries Plc Disazo dyes
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5319080A (en) 1991-10-17 1994-06-07 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US5446137A (en) 1993-12-09 1995-08-29 Syntex (U.S.A.) Inc. Oligonucleotides containing 4'-substituted nucleotides
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5466786A (en) 1989-10-24 1995-11-14 Gilead Sciences 2'modified nucleoside and nucleotide compounds
US5466677A (en) 1993-03-06 1995-11-14 Ciba-Geigy Corporation Dinucleoside phosphinates and their pharmaceutical compositions
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5514785A (en) 1990-05-11 1996-05-07 Becton Dickinson And Company Solid supports for nucleic acid hybridization assays
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5541316A (en) 1992-02-11 1996-07-30 Henkel Kommanditgesellschaft Auf Aktien Process for the production of polysaccharide-based polycarboxylates
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5552540A (en) 1987-06-24 1996-09-03 Howard Florey Institute Of Experimental Physiology And Medicine Nucleoside derivatives
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
US5567811A (en) 1990-05-03 1996-10-22 Amersham International Plc Phosphoramidite derivatives, their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5576427A (en) 1993-03-30 1996-11-19 Sterling Winthrop, Inc. Acyclic nucleoside analogs and oligonucleotide sequences containing them
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US5596086A (en) 1990-09-20 1997-01-21 Gilead Sciences, Inc. Modified internucleoside linkages having one nitrogen and two carbon atoms
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5610300A (en) 1992-07-01 1997-03-11 Ciba-Geigy Corporation Carbocyclic nucleosides containing bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5614617A (en) 1990-07-27 1997-03-25 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5639873A (en) 1992-02-05 1997-06-17 Centre National De La Recherche Scientifique (Cnrs) Oligothionucleotides
US5645620A (en) 1995-05-25 1997-07-08 Foster Wheeler Development Corp. System for separating particulates and condensable species from a gas stream
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5658873A (en) 1993-04-10 1997-08-19 Degussa Aktiengesellschaft Coated sodium percarbonate particles, a process for their production and detergent, cleaning and bleaching compositions containing them
US5663312A (en) 1993-03-31 1997-09-02 Sanofi Oligonucleotide dimers with amide linkages replacing phosphodiester linkages
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5677439A (en) 1990-08-03 1997-10-14 Sanofi Oligonucleotide analogues containing phosphate diester linkage substitutes, compositions thereof, and precursor dinucleotide analogues
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5750692A (en) 1990-01-11 1998-05-12 Isis Pharmaceuticals, Inc. Synthesis of 3-deazapurines
WO1999014226A2 (fr) 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
US6015886A (en) 1993-05-24 2000-01-18 Chemgenes Corporation Oligonucleotide phosphate esters
US6028188A (en) 1993-11-16 2000-02-22 Genta Incorporated Synthetic oligomers having chirally pure phosphonate internucleosidyl linkages mixed with non-phosphonate internucleosidyl linkages
WO2000022113A1 (fr) 1998-10-09 2000-04-20 Ingene, Inc. Synthese enzymatique d'adn simple brin
WO2000022114A1 (fr) 1998-10-09 2000-04-20 Ingene, Inc. PRODUCTION D'ADN SIMPLE BRIN $i(IN VIVO)
US6054299A (en) 1994-04-29 2000-04-25 Conrad; Charles A. Stem-loop cloning vector and method
US6124445A (en) 1994-11-23 2000-09-26 Isis Pharmaceuticals, Inc. Phosphotriester oligonucleotides, amidities and method of preparation
US6147200A (en) 1999-08-19 2000-11-14 Isis Pharmaceuticals, Inc. 2'-O-acetamido modified monomers and oligomers
US6160109A (en) 1995-10-20 2000-12-12 Isis Pharmaceuticals, Inc. Preparation of phosphorothioate and boranophosphate oligomers
US6166197A (en) 1995-03-06 2000-12-26 Isis Pharmaceuticals, Inc. Oligomeric compounds having pyrimidine nucleotide (S) with 2'and 5 substitutions
US6169170B1 (en) 1994-03-18 2001-01-02 Lynx Therapeutics, Inc. Oligonucleotide N3′→N5′Phosphoramidate Duplexes
US6172209B1 (en) 1997-02-14 2001-01-09 Isis Pharmaceuticals Inc. Aminooxy-modified oligonucleotides and methods for making same
US6222025B1 (en) 1995-03-06 2001-04-24 Isis Pharmaceuticals, Inc. Process for the synthesis of 2′-O-substituted pyrimidines and oligomeric compounds therefrom
US6235887B1 (en) 1991-11-26 2001-05-22 Isis Pharmaceuticals, Inc. Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines
US6239265B1 (en) 1990-01-11 2001-05-29 Isis Pharmaceuticals, Inc. Oligonucleotides having chiral phosphorus linkages
US6268490B1 (en) 1997-03-07 2001-07-31 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogues
US6277603B1 (en) 1991-12-24 2001-08-21 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
US6326199B1 (en) 1991-12-24 2001-12-04 Isis Pharmaceuticals, Inc. Gapped 2′ modified oligonucleotides
US6346614B1 (en) 1992-07-23 2002-02-12 Hybridon, Inc. Hybrid oligonucleotide phosphorothioates
US6410526B1 (en) 1999-06-02 2002-06-25 Merck & Co., Inc. αv integrin receptor antagonists
US6444423B1 (en) 1996-06-07 2002-09-03 Molecular Dynamics, Inc. Nucleosides comprising polydentate ligands
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
US6528640B1 (en) 1997-11-05 2003-03-04 Ribozyme Pharmaceuticals, Incorporated Synthetic ribonucleic acids with RNAse activity
US6531590B1 (en) 1998-04-24 2003-03-11 Isis Pharmaceuticals, Inc. Processes for the synthesis of oligonucleotide compounds
US6534639B1 (en) 1999-07-07 2003-03-18 Isis Pharmaceuticals, Inc. Guanidinium functionalized oligonucleotides and method/synthesis
US6608035B1 (en) 1994-10-25 2003-08-19 Hybridon, Inc. Method of down-regulating gene expression
US6617438B1 (en) 1997-11-05 2003-09-09 Sirna Therapeutics, Inc. Oligoribonucleotides with enzymatic activity
US6639062B2 (en) 1997-02-14 2003-10-28 Isis Pharmaceuticals, Inc. Aminooxy-modified nucleosidic compounds and oligomeric compounds prepared therefrom
US6670461B1 (en) 1997-09-12 2003-12-30 Exiqon A/S Oligonucleotide analogues
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
US20040171570A1 (en) 2002-11-05 2004-09-02 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US6815432B2 (en) 1995-06-07 2004-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6858225B2 (en) 1997-05-14 2005-02-22 Inex Pharmaceuticals Corporation Lipid-encapsulated polyanionic nucleic acid
US6858715B2 (en) 1999-02-04 2005-02-22 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
US6867294B1 (en) 1998-07-14 2005-03-15 Isis Pharmaceuticals, Inc. Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages
US6878805B2 (en) 2002-08-16 2005-04-12 Isis Pharmaceuticals, Inc. Peptide-conjugated oligomeric compounds
US6998484B2 (en) 2000-10-04 2006-02-14 Santaris Pharma A/S Synthesis of purine locked nucleic acid analogues
US7015315B1 (en) 1991-12-24 2006-03-21 Isis Pharmaceuticals, Inc. Gapped oligonucleotides
US7045610B2 (en) 1998-04-03 2006-05-16 Epoch Biosciences, Inc. Modified oligonucleotides for mismatch discrimination
US7053207B2 (en) 1999-05-04 2006-05-30 Exiqon A/S L-ribo-LNA analogues
US7084125B2 (en) 1999-03-18 2006-08-01 Exiqon A/S Xylo-LNA analogues
US7273933B1 (en) 1998-02-26 2007-09-25 Isis Pharmaceuticals, Inc. Methods for synthesis of oligonucleotides
US7321029B2 (en) 2000-01-21 2008-01-22 Geron Corporation 2′-arabino-fluorooligonucleotide N3′→P5′ phosphoramidates: their synthesis and use
US20080039618A1 (en) 2002-11-05 2008-02-14 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US7399845B2 (en) 2006-01-27 2008-07-15 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
US7427672B2 (en) 2003-08-28 2008-09-23 Takeshi Imanishi Artificial nucleic acids of n-o bond crosslinkage type
US7495088B1 (en) 1989-12-04 2009-02-24 Enzo Life Sciences, Inc. Modified nucleotide compounds
US7569686B1 (en) 2006-01-27 2009-08-04 Isis Pharmaceuticals, Inc. Compounds and methods for synthesis of bicyclic nucleic acid analogs
WO2010011895A1 (fr) 2008-07-25 2010-01-28 Alnylam Pharmaceuticals, Inc. Amélioration de l’activité d’extinction d’arnsi utilisant des bases universelles ou des non-appariements dans le brin sens
US7745608B2 (en) 2003-04-17 2010-06-29 Alnylam Pharmaceuticals, Inc. Modified iRNA agents
WO2011005861A1 (fr) 2009-07-07 2011-01-13 Alnylam Pharmaceuticals, Inc. Coiffes d’extrémité d’oligonucléotides
US8017762B2 (en) 2003-04-17 2011-09-13 Alnylam Pharmaceuticals, Inc. Modified iRNA agents
US8030467B2 (en) 2006-05-11 2011-10-04 Isis Pharmaceuticals, Inc. 5′-modified bicyclic nucleic acid analogs
WO2011133876A2 (fr) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. Oligonucléotides comprenant des nucléosides acycliques et abasiques, et analogues
US8058069B2 (en) 2008-04-15 2011-11-15 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
US20110313020A1 (en) 2008-12-03 2011-12-22 Marina Biotech, Inc. UsiRNA Complexes
US8101348B2 (en) 2002-07-10 2012-01-24 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. RNA-interference by single-stranded RNA molecules
US8158601B2 (en) 2009-06-10 2012-04-17 Alnylam Pharmaceuticals, Inc. Lipid formulation
US20120157511A1 (en) 2009-07-07 2012-06-21 Alnylam Pharmaceuticals, Inc. 5' phosphate mimics
US8278426B2 (en) 2007-06-08 2012-10-02 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
US8278425B2 (en) 2007-05-30 2012-10-02 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
US8278283B2 (en) 2007-07-05 2012-10-02 Isis Pharmaceuticals, Inc. 6-disubstituted or unsaturated bicyclic nucleic acid analogs
US8314227B2 (en) 2007-05-22 2012-11-20 Marina Biotech, Inc. Hydroxymethyl substituted RNA oligonucleotides and RNA complexes
US20130011922A1 (en) 2007-03-02 2013-01-10 F/K/A Mdrna, Inc. Nucleic acid compounds for inhibiting gene expression and uses thereof
WO2013036868A1 (fr) 2011-09-07 2013-03-14 Marina Biotech Inc. Synthèse et utilisations de composés acides nucléiques comportant des monomères restreints de point de vue conformationnel
US20130190383A1 (en) 2010-04-26 2013-07-25 Marina Biotech, Inc. Nucleic acid compounds with conformationally restricted monomers and uses thereof
US10000489B2 (en) 2014-09-26 2018-06-19 Glaxosmithkline Intellectual Property Development Limited Compounds αvβ6 integrin antagonists
US10023568B2 (en) 2013-03-28 2018-07-17 Glaxosmithkline Intellectual Property Development Limited Naphthyridine derivatives useful as αvβ6 integrin antagonists
WO2018136620A2 (fr) 2017-01-18 2018-07-26 Alnylam Pharmaceuticals, Inc. Lieurs clivables endosomaux
US10144733B2 (en) 2014-09-26 2018-12-04 Glaxosmithkline Intellectual Property Development Limited Naphthyridine derivatives as alpha v beta 6 integrin antagonists for the treatment of E.G. fibrotic diseases
US10487080B2 (en) 2016-03-21 2019-11-26 Glaxosmithkline Intellectual Property Development Limited Naphthyridines as integrin antagonists
WO2022147214A2 (fr) 2020-12-31 2022-07-07 Alnylam Pharmaceuticals, Inc. Promédicaments oligonucléotidiques à base de phosphate modifiés par un disulfure cyclique
WO2023064530A1 (fr) 2021-10-15 2023-04-20 Alnylam Pharmaceuticals, Inc. Compositions d'arni à administration extra-hépatique et leurs procédés d'utilisation
WO2024086633A1 (fr) 2022-10-19 2024-04-25 Arrowhead Pharmaceuticals, Inc. Agents arni servant à inhiber l'expression de la protéine kinase dm1 (dmpk), compositions de ceux-ci, et procédés d'utilisation

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102762143B1 (ko) * 2017-11-01 2025-02-07 애로우헤드 파마슈티컬스 인코포레이티드 인테그린 리간드 및 그의 용도
CN118638788A (zh) * 2017-12-06 2024-09-13 艾维迪提生物科学公司 治疗肌萎缩和强直性肌营养不良的组合物和方法
WO2019222354A1 (fr) * 2018-05-15 2019-11-21 University Of Washington Compositions et procédés pour réduire les anomalies d'épissage et traiter des troubles de dominance arn
CA3174068A1 (fr) * 2020-07-16 2022-01-20 Anastasia Khvorova Oligonucleotides conjugues permettant une administration specifique dans des tissus
MX2023002867A (es) * 2020-09-11 2023-03-23 Arrowhead Pharmaceuticals Inc Ligandos dirigidos a integrina y usos de los mismos.
US20250352672A1 (en) * 2022-05-13 2025-11-20 University Of Washington Method for treatment of myotonic dystrophy combining protein expression and rna interference vector delivery with tissue detargeting
AU2023399039A1 (en) * 2022-12-14 2025-05-22 Alnylam Pharmaceuticals, Inc. ALPHA-V BETA-6(ανβ6) INTEGRIN LIGANDS FOR EXTRAHEPATIC DELIVERY

Patent Citations (178)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US5118800A (en) 1983-12-20 1992-06-02 California Institute Of Technology Oligonucleotides possessing a primary amino group in the terminal nucleotide
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US4981957A (en) 1984-07-19 1991-01-01 Centre National De La Recherche Scientifique Oligonucleotides with modified phosphate and modified carbohydrate moieties at the respective chain termini
US5367066A (en) 1984-10-16 1994-11-22 Chiron Corporation Oligonucleotides with selectably cleavable and/or abasic sites
US4845205A (en) 1985-01-08 1989-07-04 Institut Pasteur 2,N6 -disubstituted and 2,N6 -trisubstituted adenosine-3'-phosphoramidites
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5130300A (en) 1986-03-07 1992-07-14 Monsanto Company Method for enhancing growth of mammary parenchyma
US5286717A (en) 1987-03-25 1994-02-15 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5552540A (en) 1987-06-24 1996-09-03 Howard Florey Institute Of Experimental Physiology And Medicine Nucleoside derivatives
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5453496A (en) 1988-05-26 1995-09-26 University Patents, Inc. Polynucleotide phosphorodithioate
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5175273A (en) 1988-07-01 1992-12-29 Genentech, Inc. Nucleic acid intercalating agents
US5134066A (en) 1989-08-29 1992-07-28 Monsanto Company Improved probes using nucleosides containing 3-dezauracil analogs
US5591722A (en) 1989-09-15 1997-01-07 Southern Research Institute 2'-deoxy-4'-thioribonucleosides and their antiviral activity
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5466786A (en) 1989-10-24 1995-11-14 Gilead Sciences 2'modified nucleoside and nucleotide compounds
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5466786B1 (en) 1989-10-24 1998-04-07 Gilead Sciences 2' Modified nucleoside and nucleotide compounds
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US7495088B1 (en) 1989-12-04 2009-02-24 Enzo Life Sciences, Inc. Modified nucleotide compounds
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5646265A (en) 1990-01-11 1997-07-08 Isis Pharmceuticals, Inc. Process for the preparation of 2'-O-alkyl purine phosphoramidites
US5750692A (en) 1990-01-11 1998-05-12 Isis Pharmaceuticals, Inc. Synthesis of 3-deazapurines
US6239265B1 (en) 1990-01-11 2001-05-29 Isis Pharmaceuticals, Inc. Oligonucleotides having chiral phosphorus linkages
US5681941A (en) 1990-01-11 1997-10-28 Isis Pharmaceuticals, Inc. Substituted purines and oligonucleotide cross-linking
US5459255A (en) 1990-01-11 1995-10-17 Isis Pharmaceuticals, Inc. N-2 substituted purines
US5587469A (en) 1990-01-11 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides containing N-2 substituted purines
US5670633A (en) 1990-01-11 1997-09-23 Isis Pharmaceuticals, Inc. Sugar modified oligonucleotides that detect and modulate gene expression
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5563253A (en) 1990-03-08 1996-10-08 Worcester Foundation For Biomedical Research Linear aminoalkylphosphoramidate oligonucleotide derivatives
US5536821A (en) 1990-03-08 1996-07-16 Worcester Foundation For Biomedical Research Aminoalkylphosphorothioamidate oligonucleotide deratives
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
US5567811A (en) 1990-05-03 1996-10-22 Amersham International Plc Phosphoramidite derivatives, their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides
US5514785A (en) 1990-05-11 1996-05-07 Becton Dickinson And Company Solid supports for nucleic acid hybridization assays
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5614617A (en) 1990-07-27 1997-03-25 Isis Pharmaceuticals, Inc. Nuclease resistant, pyrimidine modified oligonucleotides that detect and modulate gene expression
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5677439A (en) 1990-08-03 1997-10-14 Sanofi Oligonucleotide analogues containing phosphate diester linkage substitutes, compositions thereof, and precursor dinucleotide analogues
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
US5596086A (en) 1990-09-20 1997-01-21 Gilead Sciences, Inc. Modified internucleoside linkages having one nitrogen and two carbon atoms
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
US5177195A (en) 1991-01-08 1993-01-05 Imperial Chemical Industries Plc Disazo dyes
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5319080A (en) 1991-10-17 1994-06-07 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
US5393878A (en) 1991-10-17 1995-02-28 Ciba-Geigy Corporation Bicyclic nucleosides, oligonucleotides, process for their preparation and intermediates
US5594121A (en) 1991-11-07 1997-01-14 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified purines
US6380368B1 (en) 1991-11-26 2002-04-30 Isis Pharmaceuticals, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US6235887B1 (en) 1991-11-26 2001-05-22 Isis Pharmaceuticals, Inc. Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines
US5359044A (en) 1991-12-13 1994-10-25 Isis Pharmaceuticals Cyclobutyl oligonucleotide surrogates
US7015315B1 (en) 1991-12-24 2006-03-21 Isis Pharmaceuticals, Inc. Gapped oligonucleotides
US6277603B1 (en) 1991-12-24 2001-08-21 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
US6326199B1 (en) 1991-12-24 2001-12-04 Isis Pharmaceuticals, Inc. Gapped 2′ modified oligonucleotides
US5639873A (en) 1992-02-05 1997-06-17 Centre National De La Recherche Scientifique (Cnrs) Oligothionucleotides
US5541316A (en) 1992-02-11 1996-07-30 Henkel Kommanditgesellschaft Auf Aktien Process for the production of polysaccharide-based polycarboxylates
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US5610300A (en) 1992-07-01 1997-03-11 Ciba-Geigy Corporation Carbocyclic nucleosides containing bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US5700920A (en) 1992-07-01 1997-12-23 Novartis Corporation Carbocyclic nucleosides containing bicyclic rings, oligonucleotides therefrom, process for their preparation, their use and intermediates
US6346614B1 (en) 1992-07-23 2002-02-12 Hybridon, Inc. Hybrid oligonucleotide phosphorothioates
US6683167B2 (en) 1992-07-23 2004-01-27 University Of Massachusetts Worcester Hybrid oligonucleotide phosphorothioates
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5466677A (en) 1993-03-06 1995-11-14 Ciba-Geigy Corporation Dinucleoside phosphinates and their pharmaceutical compositions
US5576427A (en) 1993-03-30 1996-11-19 Sterling Winthrop, Inc. Acyclic nucleoside analogs and oligonucleotide sequences containing them
US5663312A (en) 1993-03-31 1997-09-02 Sanofi Oligonucleotide dimers with amide linkages replacing phosphodiester linkages
US5658873A (en) 1993-04-10 1997-08-19 Degussa Aktiengesellschaft Coated sodium percarbonate particles, a process for their production and detergent, cleaning and bleaching compositions containing them
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US6015886A (en) 1993-05-24 2000-01-18 Chemgenes Corporation Oligonucleotide phosphate esters
US5502177A (en) 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6028188A (en) 1993-11-16 2000-02-22 Genta Incorporated Synthetic oligomers having chirally pure phosphonate internucleosidyl linkages mixed with non-phosphonate internucleosidyl linkages
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5457187A (en) 1993-12-08 1995-10-10 Board Of Regents University Of Nebraska Oligonucleotides containing 5-fluorouracil
US5446137A (en) 1993-12-09 1995-08-29 Syntex (U.S.A.) Inc. Oligonucleotides containing 4'-substituted nucleotides
US5446137B1 (en) 1993-12-09 1998-10-06 Behringwerke Ag Oligonucleotides containing 4'-substituted nucleotides
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5596091A (en) 1994-03-18 1997-01-21 The Regents Of The University Of California Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides
US6169170B1 (en) 1994-03-18 2001-01-02 Lynx Therapeutics, Inc. Oligonucleotide N3′→N5′Phosphoramidate Duplexes
US5627053A (en) 1994-03-29 1997-05-06 Ribozyme Pharmaceuticals, Inc. 2'deoxy-2'-alkylnucleotide containing nucleic acid
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US6054299A (en) 1994-04-29 2000-04-25 Conrad; Charles A. Stem-loop cloning vector and method
US5525711A (en) 1994-05-18 1996-06-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pteridine nucleotide analogs as fluorescent DNA probes
US5597909A (en) 1994-08-25 1997-01-28 Chiron Corporation Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use
US6608035B1 (en) 1994-10-25 2003-08-19 Hybridon, Inc. Method of down-regulating gene expression
US6124445A (en) 1994-11-23 2000-09-26 Isis Pharmaceuticals, Inc. Phosphotriester oligonucleotides, amidities and method of preparation
US6166197A (en) 1995-03-06 2000-12-26 Isis Pharmaceuticals, Inc. Oligomeric compounds having pyrimidine nucleotide (S) with 2'and 5 substitutions
US6222025B1 (en) 1995-03-06 2001-04-24 Isis Pharmaceuticals, Inc. Process for the synthesis of 2′-O-substituted pyrimidines and oligomeric compounds therefrom
US5645620A (en) 1995-05-25 1997-07-08 Foster Wheeler Development Corp. System for separating particulates and condensable species from a gas stream
US6815432B2 (en) 1995-06-07 2004-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US6160109A (en) 1995-10-20 2000-12-12 Isis Pharmaceuticals, Inc. Preparation of phosphorothioate and boranophosphate oligomers
US6444423B1 (en) 1996-06-07 2002-09-03 Molecular Dynamics, Inc. Nucleosides comprising polydentate ligands
US6639062B2 (en) 1997-02-14 2003-10-28 Isis Pharmaceuticals, Inc. Aminooxy-modified nucleosidic compounds and oligomeric compounds prepared therefrom
US6172209B1 (en) 1997-02-14 2001-01-09 Isis Pharmaceuticals Inc. Aminooxy-modified oligonucleotides and methods for making same
US6268490B1 (en) 1997-03-07 2001-07-31 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogues
US6770748B2 (en) 1997-03-07 2004-08-03 Takeshi Imanishi Bicyclonucleoside and oligonucleotide analogue
US6858225B2 (en) 1997-05-14 2005-02-22 Inex Pharmaceuticals Corporation Lipid-encapsulated polyanionic nucleic acid
US7034133B2 (en) 1997-09-12 2006-04-25 Exiqon A/S Oligonucleotide analogues
WO1999014226A2 (fr) 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
US6794499B2 (en) 1997-09-12 2004-09-21 Exiqon A/S Oligonucleotide analogues
US6670461B1 (en) 1997-09-12 2003-12-30 Exiqon A/S Oligonucleotide analogues
US6528640B1 (en) 1997-11-05 2003-03-04 Ribozyme Pharmaceuticals, Incorporated Synthetic ribonucleic acids with RNAse activity
US6617438B1 (en) 1997-11-05 2003-09-09 Sirna Therapeutics, Inc. Oligoribonucleotides with enzymatic activity
US7273933B1 (en) 1998-02-26 2007-09-25 Isis Pharmaceuticals, Inc. Methods for synthesis of oligonucleotides
US7045610B2 (en) 1998-04-03 2006-05-16 Epoch Biosciences, Inc. Modified oligonucleotides for mismatch discrimination
US6531590B1 (en) 1998-04-24 2003-03-11 Isis Pharmaceuticals, Inc. Processes for the synthesis of oligonucleotide compounds
US6867294B1 (en) 1998-07-14 2005-03-15 Isis Pharmaceuticals, Inc. Gapped oligomers having site specific chiral phosphorothioate internucleoside linkages
USRE39464E1 (en) 1998-07-14 2007-01-09 Isis Pharmaceuticals Inc. Oligonucleolotides having site specific chiral phosphorothioate internucleoside linkages
WO2000022113A1 (fr) 1998-10-09 2000-04-20 Ingene, Inc. Synthese enzymatique d'adn simple brin
WO2000022114A1 (fr) 1998-10-09 2000-04-20 Ingene, Inc. PRODUCTION D'ADN SIMPLE BRIN $i(IN VIVO)
US6858715B2 (en) 1999-02-04 2005-02-22 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
US7041816B2 (en) 1999-02-04 2006-05-09 Isis Pharmaceuticals, Inc. Process for the synthesis of oligomeric compounds
US7084125B2 (en) 1999-03-18 2006-08-01 Exiqon A/S Xylo-LNA analogues
US7053207B2 (en) 1999-05-04 2006-05-30 Exiqon A/S L-ribo-LNA analogues
US6525191B1 (en) 1999-05-11 2003-02-25 Kanda S. Ramasamy Conformationally constrained L-nucleosides
US6410526B1 (en) 1999-06-02 2002-06-25 Merck & Co., Inc. αv integrin receptor antagonists
US6534639B1 (en) 1999-07-07 2003-03-18 Isis Pharmaceuticals, Inc. Guanidinium functionalized oligonucleotides and method/synthesis
US6147200A (en) 1999-08-19 2000-11-14 Isis Pharmaceuticals, Inc. 2'-O-acetamido modified monomers and oligomers
US7321029B2 (en) 2000-01-21 2008-01-22 Geron Corporation 2′-arabino-fluorooligonucleotide N3′→P5′ phosphoramidates: their synthesis and use
US6998484B2 (en) 2000-10-04 2006-02-14 Santaris Pharma A/S Synthesis of purine locked nucleic acid analogues
US8101348B2 (en) 2002-07-10 2012-01-24 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. RNA-interference by single-stranded RNA molecules
US6878805B2 (en) 2002-08-16 2005-04-12 Isis Pharmaceuticals, Inc. Peptide-conjugated oligomeric compounds
US20040171570A1 (en) 2002-11-05 2004-09-02 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US20080039618A1 (en) 2002-11-05 2008-02-14 Charles Allerson Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation
US7745608B2 (en) 2003-04-17 2010-06-29 Alnylam Pharmaceuticals, Inc. Modified iRNA agents
US8017762B2 (en) 2003-04-17 2011-09-13 Alnylam Pharmaceuticals, Inc. Modified iRNA agents
US7427672B2 (en) 2003-08-28 2008-09-23 Takeshi Imanishi Artificial nucleic acids of n-o bond crosslinkage type
US20090012281A1 (en) 2006-01-27 2009-01-08 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
US7569686B1 (en) 2006-01-27 2009-08-04 Isis Pharmaceuticals, Inc. Compounds and methods for synthesis of bicyclic nucleic acid analogs
US8022193B2 (en) 2006-01-27 2011-09-20 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
US7741457B2 (en) 2006-01-27 2010-06-22 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
US7399845B2 (en) 2006-01-27 2008-07-15 Isis Pharmaceuticals, Inc. 6-modified bicyclic nucleic acid analogs
US8030467B2 (en) 2006-05-11 2011-10-04 Isis Pharmaceuticals, Inc. 5′-modified bicyclic nucleic acid analogs
US20130011922A1 (en) 2007-03-02 2013-01-10 F/K/A Mdrna, Inc. Nucleic acid compounds for inhibiting gene expression and uses thereof
US20130096289A1 (en) 2007-05-22 2013-04-18 Marina Biotech, Inc. Hydroxymethyl substituted rna oligonucleotides and rna complexes
US8314227B2 (en) 2007-05-22 2012-11-20 Marina Biotech, Inc. Hydroxymethyl substituted RNA oligonucleotides and RNA complexes
US8278425B2 (en) 2007-05-30 2012-10-02 Isis Pharmaceuticals, Inc. N-substituted-aminomethylene bridged bicyclic nucleic acid analogs
US8278426B2 (en) 2007-06-08 2012-10-02 Isis Pharmaceuticals, Inc. Carbocyclic bicyclic nucleic acid analogs
US8278283B2 (en) 2007-07-05 2012-10-02 Isis Pharmaceuticals, Inc. 6-disubstituted or unsaturated bicyclic nucleic acid analogs
US8058069B2 (en) 2008-04-15 2011-11-15 Protiva Biotherapeutics, Inc. Lipid formulations for nucleic acid delivery
WO2010011895A1 (fr) 2008-07-25 2010-01-28 Alnylam Pharmaceuticals, Inc. Amélioration de l’activité d’extinction d’arnsi utilisant des bases universelles ou des non-appariements dans le brin sens
US20110313020A1 (en) 2008-12-03 2011-12-22 Marina Biotech, Inc. UsiRNA Complexes
US8158601B2 (en) 2009-06-10 2012-04-17 Alnylam Pharmaceuticals, Inc. Lipid formulation
US20120157511A1 (en) 2009-07-07 2012-06-21 Alnylam Pharmaceuticals, Inc. 5' phosphate mimics
WO2011005861A1 (fr) 2009-07-07 2011-01-13 Alnylam Pharmaceuticals, Inc. Coiffes d’extrémité d’oligonucléotides
WO2011133876A2 (fr) 2010-04-22 2011-10-27 Alnylam Pharmaceuticals, Inc. Oligonucléotides comprenant des nucléosides acycliques et abasiques, et analogues
US20130190383A1 (en) 2010-04-26 2013-07-25 Marina Biotech, Inc. Nucleic acid compounds with conformationally restricted monomers and uses thereof
WO2013036868A1 (fr) 2011-09-07 2013-03-14 Marina Biotech Inc. Synthèse et utilisations de composés acides nucléiques comportant des monomères restreints de point de vue conformationnel
US10450312B2 (en) 2013-03-28 2019-10-22 Glaxosmithkline Intellectual Property Development Limited Naphthyridine derivatives useful as alpha-V-beta-6 integrin antagonists
US10023568B2 (en) 2013-03-28 2018-07-17 Glaxosmithkline Intellectual Property Development Limited Naphthyridine derivatives useful as αvβ6 integrin antagonists
US10144733B2 (en) 2014-09-26 2018-12-04 Glaxosmithkline Intellectual Property Development Limited Naphthyridine derivatives as alpha v beta 6 integrin antagonists for the treatment of E.G. fibrotic diseases
US10000489B2 (en) 2014-09-26 2018-06-19 Glaxosmithkline Intellectual Property Development Limited Compounds αvβ6 integrin antagonists
US10513517B2 (en) 2014-09-26 2019-12-24 Glaxosmithkline Intellectual Property Development Limited Naphthyridine derivatives as alpha V beta 6 integrin antagonists for the treatment of E.G. fibrotic diseases
US10487080B2 (en) 2016-03-21 2019-11-26 Glaxosmithkline Intellectual Property Development Limited Naphthyridines as integrin antagonists
WO2018136620A2 (fr) 2017-01-18 2018-07-26 Alnylam Pharmaceuticals, Inc. Lieurs clivables endosomaux
WO2022147214A2 (fr) 2020-12-31 2022-07-07 Alnylam Pharmaceuticals, Inc. Promédicaments oligonucléotidiques à base de phosphate modifiés par un disulfure cyclique
WO2023064530A1 (fr) 2021-10-15 2023-04-20 Alnylam Pharmaceuticals, Inc. Compositions d'arni à administration extra-hépatique et leurs procédés d'utilisation
WO2024086633A1 (fr) 2022-10-19 2024-04-25 Arrowhead Pharmaceuticals, Inc. Agents arni servant à inhiber l'expression de la protéine kinase dm1 (dmpk), compositions de ceux-ci, et procédés d'utilisation

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. XM 045381179.1
"The Concise Encyclopedia Of Polymer Science And Engineering", 1990, JOHN WILEY & SONS, pages: 858 - 859
CHATTOPADHYAYA ET AL., J. ORG. CHEM., vol. 74, 2009, pages 118 - 134
CHURANA, RNA, vol. 14, 2007, pages 1714 - 1719
COUTURE, A ET AL., TIG, vol. 12, 1996, pages 5 - 10
DIAS, N. ET AL., MOL CANCER THER, vol. 1, 2002, pages 347 - 355
ELBASHIR ET AL., EMBO, vol. 20, 2001, pages 6877 - 6888
ELMEN, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 33, no. 1, 2005, pages 439 - 447
ENGLISCH ET AL., ANGEWANDTE CHEMIE, vol. 30, 1991, pages 613
FLUITER ET AL., MOL. BIOSYST., vol. 10, 2009, pages 1039
GASSMANN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 1292
GRUNWELLER, A. ET AL., NUCLEIC ACIDS RESEARCH, vol. 31, no. 12, 2003, pages 3185 - 3193
KIM ET AL., NAT BIOTECH, vol. 23, 2005, pages 222 - 226
LIMA ET AL., CELL, vol. 150, 2012, pages 883 - 894
MARTIN ET AL., HELV. CHIM. ACTA, vol. 78, 1995, pages 486 - 504
MIKHAILOV, TETRAHEDRON LETTERS, vol. 26, no. 17, 1985, pages 2059
MOOK, OR. ET AL., MOL CANC THER, vol. 6, no. 3, 2007, pages 833 - 843
NIELSEN ET AL., SCIENCE, vol. 254, 1991, pages 1497 - 1500
NUC. ACIDS SYMP. SERIES, vol. 52, 2008, pages 133 - 134
PATRI ET AL., CURR. OPIN. CURR. BIOL., vol. 6, 2002, pages 466 - 471
QUINTANA ET AL., PHARM RES., vol. 19, 2002, pages 1310 - 1316
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SANGHVI, Y S.: "dsRNA Research and Applications", 1993, CRC PRESS, pages: 276 - 278
T. W. GREENEP. G. M. WUTS: "Protecting Groups in Organic Synthesis", 1999, JOHN WILEY & SONS
TETKO ET AL., J. CHEM. INF. COMPUT. SCI., vol. 41, 2001, pages 1407 - 21

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