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WO2025064660A2 - Activin a receptor type 1c (acvr1c) irna compositions and methods of use thereof - Google Patents

Activin a receptor type 1c (acvr1c) irna compositions and methods of use thereof Download PDF

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Publication number
WO2025064660A2
WO2025064660A2 PCT/US2024/047464 US2024047464W WO2025064660A2 WO 2025064660 A2 WO2025064660 A2 WO 2025064660A2 US 2024047464 W US2024047464 W US 2024047464W WO 2025064660 A2 WO2025064660 A2 WO 2025064660A2
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nucleotide
dsrna agent
antisense strand
seq
nucleotides
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WO2025064660A3 (en
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Elane FISHILEVICH
Aditi Dubey
James D. MCININCH
Mark K. SCHLEGEL
Adam CASTORENO
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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
    • C12N15/1138—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 receptors or cell surface proteins
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • ACVR1C functions to antagonize catabolic pathways of lipolysis and amino acid degradation to facilitate the preservation and accumulation of energy stores.
  • a polymorphism in ACVR1C has been found to be associated with increased risk of metabolic syndrome in Chinese females and may be involved in cardiovascular remodeling in patients with metabolic syndrome (Zhang, W et al. Arq Bras Cardiol.2013: 101(2):134-140). Additionally, variants predicted to lead to loss of ACVR1C gene function are thought to influence body fat distribution and protect against type 2 diabetes (Emdin CA et al. Diabetes.2019: 68(1):226-234; Justice AE et al.
  • ACVR1C dysfunction due to a nonsense mutation
  • ACVR1C activation inhibited lipolysis by suppressing the expression of adipose lipases (Guo T et al. eLife 2014.1-18 (DOI: 10.7554/eLife.03245; Adam RC et al. Proc. Natl. Acad. Sci. U.S.A.2023.120(32)).
  • the ACVR1C 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 an ACVR1C gene, and/or for treating a subject who would benefit from inhibiting or reducing the expression of an ACVR1C gene, e.g., a subject suffering or prone to suffering from an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, diabetes, hypertension, and/or cardiovascular disease.
  • an ACVR1C-associated disorder e.g., a metabolic disorder, e.g., metabolic syndrome, diabetes, hypertension, and/or cardiovascular disease.
  • the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell
  • dsRNA double stranded ribonucleic acid
  • ACVR1C activin A receptor type 1C
  • the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region
  • the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from the nucleotide sequence of SEQ ID NO:2 and wherein one or more C 22 hydrocarbon chains are conjugated to the sense strand.
  • the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12
  • the disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte and/or a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from
  • the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte and/or a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding ACVR1C, and wherein the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.
  • dsRNA double stranded ribonucleic acid
  • ACVR1C activin A receptor type 1C
  • the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte and/or a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the sense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.
  • these dsRNA agents further comprise one or more C 22 hydrocarbon chains conjugated to one or more positions, e.g., internal positions, on at least one strand of the dsRNA agent.
  • the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the sense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing
  • the dsRNA agent comprises a sense strand comprising a contiguous nucleotide sequence which has at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, nucleotide sequence identity over its entire length to any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising a contiguous nucleotide sequence which has at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, nucleotide sequence identity over its entire length to any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12.
  • the dsRNA agent comprises a sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than three nucleotides from any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides differing by no more than three nucleotides from any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12.
  • the dsRNA agent comprises a sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than two nucleotides from any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, or 23 contiguous nucleotides differing by no more than two nucleotides from any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12.
  • the dsRNA agent comprises a sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than one nucleotide from any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides differing by no more than one nucleotide from any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12.
  • the dsRNA agent comprises a sense strand comprising or consisting of a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising or consisting of a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12.
  • the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell
  • dsRNA double stranded ribonucleic acid
  • ACVR1C activin A receptor type 1C
  • the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region
  • the antisense strand comprises a region of complementarity to an mRNA encoding ACVR1C
  • the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, Docket No.: 121301-22520 ALN-511-WO contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12.
  • the sense strand comprises at least 15, e.g., 15, 16, 17, 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 2-5, 7, 8, 11, and 12, and the antisense strand comprises at least 15, e.g.,15, 16, 17, 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 2-5, 7, 8, 11, and 12.
  • the antisense strand comprises at least 15, e.g.,15, 16, 17, 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 of a duplex selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, and AD-2508176.
  • the sense strand and the antisense strand comprises at least 15, e.g.,15, 16, 17, 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 of a duplex selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, or AD-2508176.
  • the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell
  • dsRNA double stranded ribonucleic acid
  • ACVR1C activin A receptor type 1C
  • the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region
  • the antisense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the antisense strand nucleotide sequences selected from the group consisting of (a) 5’-UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (
  • the sense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides and the antisense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) 5’- AAGCCUAAUGAUGAUAAUUAA-3’ of SEQ ID NO: 14909 and 5’- UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-AUAUCUCAACUUUGUGUCAAA-3’ of SEQ ID NO: 14910 and 5’- UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID
  • the dsRNA agent comprises at least one modified nucleotide. In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.
  • all of the nucleotides of the sense strand are modified nucleotides; all of the nucleotides of the antisense strand are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.
  • At least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3’-terminal deoxythimidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C-alkyl-modified nucleotide, 2’-hydroxly-modified nucleotide, a 2’-methoxyethyl modified nucleotide, a 2’-O-alky
  • At least one of the modified nucleotides is selected from the group 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, a 2'-deoxy-modified nucleotide, a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, a nucleotide with a 2’ phosphate, e.g., G2p, C2p, A2p or U2p, a nucleotide comprising a phosphorothioate group, and a vinyl-phosphonate nucleotide; and combinations thereof.
  • GNA glycol modified nucleotide
  • At least one of the modified nucleotides is a nucleotide with a thermally destabilizing nucleotide modification.
  • the thermally destabilizing nucleotide modification is 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).
  • the modified nucleotide comprises a short sequence of 3’-terminal deoxythimidine nucleotides (dT).
  • the dsRNA agents further comprise a phosphate or phosphate mimic at the 5’-end of the antisense strand.
  • phosphate mimic is a 5’-vinyl phosphonate (VP).
  • the 5’-end of the antisense strand of the dsRNA agent does not contain a 5’-vinyl phosphonate (VP).
  • the dsRNA agent further comprises at least one terminal, chiral phosphorus atom.
  • a site specific, chiral modification to the internucleotide linkage may occur at the 5’ end, 3’ end, or both the 5’ end and 3’ end of a strand. This is being referred to herein as a “terminal” chiral modification.
  • the terminal modification may occur at a 3’ or 5’ terminal position in a terminal region, e.g., at a position on a terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a strand.
  • a chiral modification may occur on the sense strand, antisense strand, or both the sense strand and antisense strand.
  • Each of the chiral pure phosphorus atoms may be in either Rp configuration or Sp configuration, and combination thereof.
  • the dsRNA agent further comprises a terminal, chiral modification occuring at the first internucleotide linkage at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.
  • the dsRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
  • the dsRNA agent further comprises a terminal, chiral modification occuring at the first, second, and third internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
  • the dsRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having Docket No.: 121301-22520 ALN-511-WO the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the third internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
  • the dsRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first, and second internucleotide linkages at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
  • 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, [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, piperid
  • the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
  • the phosphorothioate or methylphosphonate internucleotide linkage is at the 3’-terminus of one strand, e.g., the antisense strand or the sense strand.
  • the phosphorothioate or methylphosphonate internucleotide linkage is at the 5’-terminus of one strand, e.g., the antisense strand or the sense strand.
  • the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5’- and 3’-terminus of one strand.
  • the strand is the antisense strand.
  • the base pair at the 1 position of the 5 ⁇ -end of the antisense strand of the duplex is an AU base pair.
  • 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 region of complementarity may be at least 17 nucleotides in length; between 19 and 23 nucleotides in length; or 19 nucleotides in length.
  • at least one strand comprises a 3’ overhang of at least 1 nucleotide.
  • at least one strand comprises a 3’ overhang of at least 2 nucleotides.
  • one or more C 22 hydrocarbon chains is conjugated to one or more internal positions on at least one strand of the dsRNA agent.
  • the lipophilicity of the one or more C 22 hydrocarbon chain measured by octanol-water partition coefficient, logK ow , exceeds 0.
  • the lipophilic moiety may possess a logK ow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10.
  • the hydrophobicity of the dsRNA agent measured by the unbound fraction in the plasma protein binding assay of the dsRNA agent, exceeds 0.2.
  • the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein.
  • the hydrophobicity of the dsRNA agent measured by fraction of unbound dsRNA 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.
  • the C 22 hydrocarbon chain may be saturated or unsaturated.
  • the C 22 hydrocarbon chain may be linear or branched
  • the internal positions include all positions except the three terminal positions from each end of the at least one strand.
  • the internal positions exclude a cleavage site region of the sense strand.
  • the internal positions exclude positions 9-12 or positions 11-13, counting from the 5’-end of the sense strand.
  • the internal positions exclude a cleavage site region of the antisense strand. In some embodiments, the internal positions exclude positions 12-14, counting from the 5’-end of the antisense strand. In some embodiments, the one or more C 22 hydrocarbon chains are conjugated to 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 some embodiments, the one or more C 22 hydrocarbon chains are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’-end of each strand.
  • 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., an ACVR1C nucleotide sequence, as defined herein.
  • a target sequence e.g., an ACVR1C nucleotide sequence
  • the mismatches can be in the internal or terminal regions of the Docket No.: 121301-22520 ALN-511-WO molecule.
  • the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5’- or 3’-end of the iRNA.
  • 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.
  • 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 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.
  • a “subject” is an animal, such as a mammal, including a primate (such as a 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 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
  • treating refers to a beneficial or desired result, such as reducing at least one sign or symptom associated with ACVR1C expression or ALK7 protein production, e.g., an ACVR1C-associated disease, e.g., a metabolic disorder, in a subject.
  • Treatment Docket No.: 121301-22520 ALN-511-WO also includes a reduction of one or more sign or symptoms associated with unwanted ACVR1C expression; diminishing the extent of unwanted ACVR1C activation or stabilization; amelioration or palliation of unwanted ACVR1C activation or stabilization.
  • Treatment can also mean prolonging survival as compared to expected survival in the absence of treatment.
  • Non-limiting examples of metabolic Docket No.: 121301-22520 ALN-511-WO diseases include disorders of carbohydrates, e.g., diabetes, type I diabetes, type II diabetes, galactosemia, hereditary fructose intolerance, fructose 1,6-diphosphatase deficiency, glycogen storage disorders, congenital disorders of glycosylation, insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism; disorders of amino acid metabolism, e.g., maple syrup urine disease (MSUD), or homocystinuria; disorder of organic acid metabolism, e.g.,methylmalonic aciduria, 3-methylglutaconic aciduria -Barth syndrome, glutaric aciduria or 2-hydroxyglutaric aciduria – D and L forms; disorders of fatty acid beta-oxidation, e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD), long-chain 3-hydroxyacyl
  • Type I diabetes is also referred to as insulin-dependent diabetes mellitus, IDDM, and juvenile onset diabetes. People with type I diabetes (insulin-dependent diabetes) produce little or no insulin at all. Although about 6 percent of the United States population has some form of diabetes, only about 10 percent of all diabetics have type I disorder. Most people who have type I diabetes develop the disorder before age ,)( Gc[Q * PUMNQ ⁇ Q] ⁇ Q[ ⁇ Q]QY ⁇ ] ⁇ TQ ⁇ Q]_W ⁇ ZR M [ ⁇ ZS ⁇ Q]]U ⁇ Q M_ ⁇ ZUXX_YQ PQ] ⁇ _O ⁇ UZY ZR ⁇ TQ [MYO ⁇ QM ⁇ UO q' cells with subsequent insulin deficiency.
  • Obesity is a risk factor for type II diabetes, and 80 to 90 percent of the people with this disorder are obese.
  • diabetes includes pre-diabetes. “Pre-diabetes” refers to one or more early diabetic conditions including impaired glucose utilization, abnormal or impaired fasting glucose levels, impaired glucose tolerance, impaired insulin sensitivity and insulin resistance. Prediabetes is a major risk factor for the development of type 2 diabetes mellitus, cardiovascular disease and mortality.
  • diabetes includes secondary or other specific types of diabetes, e.g., includes monogenic defects of beta cell function, genetic defects of insulin action, exocrine pancreatic disease, endocrinopathies, drug/chemical induced, infectious, and uncommon immune-mediated and genetic syndromes associated with diabetes.
  • Exemplary uncommon immune-mediated and genetic syndromes associated with diabetes may include, but are not limited to, “Stiff-man” syndrome, anti-insulin receptor antibodies, Down syndrome, Klinefelter syndrome, Turner syndrome, Wolfram syndrome, Friedreich ataxia, Huntington chorea, Laurence-Moon-Biedl syndrome,Myotonic dystrophy, Porphyria, or Prader-Willi syndrome.Diabetes can be diagnosed by the administration of a glucose tolerance test. Clinically, diabetes is often divided into several basic categories.
  • autoimmune diabetes mellitus non-insulin-dependent diabetes mellitus (type 1 NDDM), insulin- dependent diabetes mellitus (type 2 IDDM), non-autoimmune diabetes mellitus, non-insulin- dependent diabetes mellitus (type 2 NIDDM), and maturity-onset diabetes of the young (MODY).
  • a further category often referred to as secondary, refers to diabetes brought about by some identifiable condition which causes or allows a diabetic syndrome to develop. Examples of secondary categories include, diabetes caused by pancreatic disease, hormonal abnormalities, drug- or chemical-induced diabetes, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes.
  • a metabolic disorder is a lipid metabolism disorder.
  • lipid metabolism disorder or “disorder of lipid metabolism” refers to any disorder associated with or caused by a disturbance in lipid metabolism. This term also includes any disorder, disease or condition that can lead to hyperlipidemia, or condition characterized by abnormal elevation of levels of any or all lipids and/or lipoproteins in the blood.
  • This term refers to an inherited disorder, such as familial hypertriglyceridemia, familial partial lipodystrophy type (FPLD) (e.g., familial partial lipodystrophy type 1 (FPLD1) or Köbberling type, FPLD 2 or Dunnigan type, FPLD 3 or PPARG- related FPLD, FPLD 4 or PLIN1-related FPLD, FPLD 5 or CIDEC-related FPLD, FPLD 6 or LIPE- related FPLD, CAV1-related FPLD, ADRA2A-related FPLD, AKT2-related FPLD, Progeroid syndromes, MAD type A, Werner syndrome, MDPL syndrome, Complex genetic syndromes with partial lipodystrophy, Bloom syndrome, SHORT syndrome, Autoinflammatory syndromes, or CANDLE syndrome), or an induced or acquired disorder, such as a disorder induced or acquired as a result of a disease, disorder or condition (e.g., renal failure), a diet, or intake of certain drugs (e.
  • disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, PU_ ⁇ Q ⁇ UO'UYP_OQP Tc[Q ⁇ USWcOQ ⁇ UPQXUM& MWOZTZW'UYP_OQP Tc[Q ⁇ USWcOQ ⁇ UPQXUM& q'MP ⁇ QYQ ⁇ SUO NWZOVUYS agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, post-menopausal lipid Docket No.: 121301-22520 ALN-511-WO metabolism disorder, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertrig
  • Cardiovascular diseases are also considered “metabolic disorders”, as defined herein. These diseases may include coronary artery disease (also called ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular diseases, and stroke. Kidney diseases are also considered “metabolic disorders”, as defined herein. Such diseases may include chronic kidney disease, diabetic nephrophathy, diabetic kidney disease, or gout. Disorders related to body weight are also considered “metabolic disorders”, as defined herein. Such disorders may include obesity, hypo-metabolic states, hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight regain following weight loss. Blood sugar disorders are further considered “metabolic disorders”, as defined herein.
  • Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome related to insulin resistance.
  • Other exemplary disorders of metabolic disorders may also include renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease.
  • a metabolic disorder is primary hypertension. “Primary hypertension” is a result of environmental or genetic causes (e.g., a result of no obvious underlying medical cause).
  • a metabolic disorder disorder is secondary hypertension.
  • a metabolic disorder is resistant hypertension.
  • “Resistant hypertension” is blood pressure that remains above goal (e.g., above 130 mm Hg systolic or above 90 diastolic) in spite of concurrent use of three antihypertensive agents of different classes, one of which is a thiazide diuretic. Subjects whose blood pressure is controlled with four or more medications are also considered to have resistant hypertension. Docket No.: 121301-22520 ALN-511-WO Additional diseases or conditions related to metabolic disorders that would be apparent to the skilled artisan and are within the scope of this disclosure.
  • 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 RNAi agent that, when administered to a subject having an ACVR1C-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.
  • samples may be derived from the adipose (e.g., certain segments of adipose tissue or certain types of cells in the adipose, such as, e.g., adipocytes).
  • samples may be derived from subcutaneous white adipose tissue (scWAT) (e.g., abdominal or inguinal), visceral white adipose tissue (VWAT) (e.g., mesenteric, gonadal, perirenal), or brown adipose tissue (BAT) (e.g., interscapular, periaortic).
  • scWAT subcutaneous white adipose tissue
  • VWAT visceral white adipose tissue
  • BAT brown adipose tissue
  • a “sample derived from a subject” refers to urine obtained from the subject.
  • a “sample derived from a subject” can refer to blood or blood derived serum or plasma from the subject.
  • the iRNA includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ACVR1C gene in a cell (e.g., an adipocyte), such as a cell within a subject, e.g., a mammal, such as a human susceptible to developing an ACVR1C-associated disease, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight.
  • dsRNA double stranded ribonucleic acid
  • 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 an ACVR1C 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 ACVR1C gene, the iRNA inhibits the expression of the ACVR1C 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 or LCMS, using, for example, western blotting or flow cytometric techniques.
  • inhibition of expression is determined by the qPCR method 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 rodent expressing the human gene e.g., a mouse or an AAV-infected mouse expressing the human target gene
  • 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 strand) 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 an ACVR1C gene.
  • the other strand includes a 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- 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, 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- 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- 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 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, Docket No.: 121301-22520 ALN-511-WO 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 dsRNA is not a naturally occurring miRNA.
  • an iRNA agent useful to target ACVR1C 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 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.
  • 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.
  • 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. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared.
  • 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 provided in any one of Tables 2-5, 7, 8, 11, and 12, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 2-5, 7, 8, 11, and 12.
  • 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 oligonucleotide is described as the sense strand in any one of Tables 2-5, 7, 8, 11, and 12, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-5, 7, 8, 11, and 12.
  • the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.
  • the sense or antisense strands are selected from the sense or antisense strand of any one of duplexes AD-1735894, AD-1735913, AD-2410459, AD-2410460, AD-2410461, Docket No.: 121301-22520 ALN-511-WO AD-2410462, AD-2509512, AD-2640052, AD-2640053, AD-2640060, AD-2509191, AD-2509494, AD-2509477, and AD-2508176.
  • the RNA of the iRNA of the disclosure e.g., a dsRNA of the disclosure
  • the disclosure encompasses dsRNA of Tables 2-5, 7, 8, 11, and 12 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 (Elbashir et al., EMBO 2001, 20:6877-6888).
  • 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 least one strand of a length of minimally 21 nucleotides.
  • 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 2-5, 7, 8, 11, and 12, and differing in their ability to inhibit the expression of an ACVR1C 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.
  • RNAs provided in Tables 2-5, 7, 8, 11, and 12 identify a site(s) in an ACVR1C transcript that is susceptible to RISC-mediated cleavage.
  • 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 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 2-5, 7, 8, 11, and 12 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ACVR1C gene.
  • the RNA of the iRNA of the disclosure e.g., a dsRNA
  • the RNA of an iRNA 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.
  • the dsRNA agents of the disclosure comprise at least one nucleic acid modification described herein.
  • the modification can be present in one of the RNA molecules.
  • the dsRNA agents of the disclosure comprise one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand and do not comprise additional chemical modifications known in the art and described herein, in the remaining positions of the sense and anti-sense strands.
  • the dsRNA agents of the disclosure comprise one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand, and comprise at least one additional nucleic acid modification described herein.
  • 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 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; 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 bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleot
  • RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages.
  • RNAs having 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 RNAi agent will have a phosphorus atom in its internucleoside backbone.
  • Nucleobase Modifications The naturally occurring base portion of a nucleoside is typically a heterocyclic base.
  • oligonucleotides those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the ZWUSZY_OWQZ ⁇ UPQ( GTQ YM ⁇ _ ⁇ MWWc ZOO_ ⁇ UYS WUYVMSQ Z ⁇ NMOVNZYQ ZR EB5 MYP ZR 8B5 U] M ,o ⁇ Z .o phosphodiester linkage.
  • nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U)
  • A purine nucleobase
  • G guanine
  • T pyrimidine nucleobase
  • T thymine
  • C cytosine
  • U uracil
  • modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein.
  • the unmodified or natural nucleobases can be modified or replaced to provide iRNAs having improved properties.
  • nuclease resistant oligonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the oligomer modifications described herein.
  • nucleobases e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine
  • substituted or modified analogs of any of the above bases and “universal bases” can be employed.
  • the nucleotide is said to comprise a modified nucleobase and/or a nucleobase modification herein.
  • Modified nucleobase and/or nucleobase modifications also include natural, non-natural and universal bases, which comprise conjugated moieties, e.g. a ligand described herein.
  • Conjugate moieties for conjugation with nucleobases include cationic amino groups which can be conjugated to the nucleobase via an appropriate alkyl, alkenyl or a linker with an amide linkage.
  • An oligomeric compound described herein can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • unmodified or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2- (aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine,
  • a universal nucleobase is any nucleobase that can base pair with all of the four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the iRNA duplex.
  • Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro- 6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9- methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7- azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, napthal
  • nucleobases include those disclosed in U.S. Pat. No.3,687,808; those disclosed in International Application No. PCT/US09/038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P.Ed.
  • a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G- clamp.
  • nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic.
  • DsRNA agent of the disclosures provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomer, including a nucleoside or nucleotide, having a modified sugar moiety.
  • the furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non- geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid.
  • each of the linkers of the LNA compounds is, independently, — [C(R1)(R2)]n-, —[C(R1)(R2)]n-O—, —C(R1R2)-N(R1)-O— or —C(R1R2)-O—N(R1)-.
  • the linkage can be a methylene (—CH 2 -) S ⁇ Z_[ N ⁇ UPSUYS ⁇ TQ +o ZbcSQY M ⁇ ZX MYP ⁇ TQ -o OM ⁇ NZY M ⁇ ZX& RZ ⁇ aTUOT ⁇ TQ ⁇ Q ⁇ X XQ ⁇ TcWQYQZbc #-o'7 ⁇ 2 - C'+o$ @B5 U] _]QP RZ ⁇ ⁇ TQ NUOcOWUO XZUQ ⁇ c3 UY ⁇ TQ OM]Q ZR MY Q ⁇ TcWQYQ S ⁇ Z_[ UY ⁇ TU] [Z]U ⁇ UZY& ⁇ TQ ⁇ Q ⁇ X Q ⁇ TcWQYQZbc #-o'7 ⁇ 2 CH 2 'C'+o$ @B5 U] _]QP #FUYST et al., Chem.
  • a representative list of modified sugars includes but is not limited to bicyclic modified sugars, including methyleneoxy #-o'7 ⁇ 2 'C'+o$ @B5 MYP Q ⁇ TcWQYQZbc #-o'#7 ⁇ 2 ) 2 'C'+o N ⁇ UPSQ$ 9B53 ]_N] ⁇ U ⁇ _ ⁇ QP ]_SM ⁇ ]& Q][QOUMWWc +o' ]_N] ⁇ U ⁇ _ ⁇ QP ]_SM ⁇ ] TM ⁇ UYS M +o':& +o'C7 ⁇ 3 Z ⁇ M +o'C#7 ⁇ 2 ) 2 -OCH 3 ]_N] ⁇ U ⁇ _QY ⁇ S ⁇ Z_[3 MYP -o' ⁇ TUZ modified sugars.
  • an oligomeric compound can include one or more monomers containing e.g., arabinose, as the sugar.
  • the monomer can have an alpha linkage at the 1’ position on the sugar, e.g., alpha-nucleosides.
  • the monomer can also have the opposite configuration at the 4’-position, e.g., C5’ and H4’ or substituents replacing them are interchanged with each other. When the C5’ and H4’ or substituents replacing them are interchanged with each other, the sugar is said to be modified at the 4’ position.
  • DsRNA agent of the disclosures disclosed herein can also include abasic sugars, i.e., a sugar which lack a nucleobase at C-1 ⁇ or has other chemical groups in place of a nucleobase at C1’. See for example U.S. Pat. No. 5,998,203, content of which is herein incorporated in its entirety. These abasic sugars can also be further containing modifications at one or more of the constituent sugar atoms. DsRNA agent of the disclosures can also contain one or more sugars that are the L isomer, e.g. L- nucleosides. Modification to the sugar group can also include replacement of the 4’-O with a sulfur, optionally substituted nitrogen or CH 2 group.
  • abasic sugars i.e., a sugar which lack a nucleobase at C-1 ⁇ or has other chemical groups in place of a nucleobase at C1’. See for example U.S. Pat. No. 5,998,203, content of which is herein
  • linkage between C1’ and Y_OWQZNM]Q U] UY l OZYRUS_ ⁇ M ⁇ UZY( Sugar modifications can also include acyclic nucleotides, wherein a C-C bonds between ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, C1’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are or in combination absent from the nucleotide.
  • ribose carbons e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’
  • nucleotide B is a modified or unmodified nucleobase
  • R 1 and R 2 independently are H, halogen, OR 3 , or alkyl
  • R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • xylose configuration refers to the placement of a substituent on the C3’ of ribose in the same configuration as the 3’-OH is in the xylose sugar.
  • C4’ and C5’ together form an optionally substituted heterocyclic, preferably comprising at least one -PX(Y)-, wherein X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino, where M is independently for each occurrence an alki metal or transition metal with an overall charge of +1; and Y is O, S, or NR’, where R’ is hydrogen, optionally substituted aliphatic.
  • LNA's include nucleoside having the formula: wherein: Docket No.: 121301-22520 ALN-511-WO Bx is a heterocyclic base moiety; T 1 is H or a hydroxyl protecting group; T 2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z is C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 1 -C 6 alkyl, substituted C 2 - C 6 alkenyl, substituted C 2 -C 6 alkynyl, acyl, substituted acyl, or substituted amide.
  • each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, F>*& B,& C7#nI$>*& MYP B>,7#nI$B>*>+& aTQ ⁇ QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY ⁇ Wc& ⁇ & 7 1 -C 6 alkyl, or substituted C 1 -C 6 alkyl and X is O or NJ1.
  • the Z group is C 1 -C 6 alkyl substituted with one or more Xx, wherein QMOT Ib U] UYPQ[QYPQY ⁇ Wc C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z ⁇ 7B3 wherein each J1, J2 and J3 is, independently, H or C 1 -C 6 alkyl, and X is O, S or NJ1.
  • the Z group is C 1 -C 6 alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—), substituted alkoxy or azido.
  • Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—), substituted alkoxy or azido.
  • the Z group is —CH 2 Xx, wherein Xx is OJ1, NJ1J2, SJ1, N3, C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z ⁇ 7B3 aTQ ⁇ QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY ⁇ Wc& ⁇ or C 1 -C 6 alkyl, and X is O, S or NJ1.
  • the Z group is —CH 2 Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—) or azido.
  • each T 1 and T 2 is a hydroxyl protecting group.
  • a preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9- (p-methoxyphenyl)xanthine-9-yl (MOX).
  • T 1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is T 1 U] -&-o'PUXQ ⁇ TZbc ⁇ U ⁇ cW( Docket No.: 121301-22520 ALN-511-WO
  • T 2 is a reactive phosphorus group wherein preferred reactive phosphorus groups include diisopropylcyanoethoxy phosphoramidite and H-phosphonate.
  • T 1 U] -&-o'PUXQ ⁇ TZbc ⁇ U ⁇ cW MYP G 2 is diisopropylcyanoethoxy phosphoramidite.
  • the compounds of the disclosure comprise at least one monomer of the formula: or of the formula: or of the formula: T3 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound;
  • T4 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group
  • each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, Docket No.: 121301-22520 ALN-511-WO F>*& B,& C7#nI$>*& MYP B>,7#nI$B>*>+& aTQ ⁇ QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY ⁇ Wc& ⁇ Z ⁇ 7 1 -C 6 alkyl, and X is O or NJ1.
  • at least one Z is C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl.
  • each Z is, independently, C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl. In certain embodiments, at least one Z is C 1 -C 6 alkyl. In certain embodiments, each Z is, independently, C 1 -C 6 alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C 1 -C 6 alkyl. In certain embodiments, each Z is, independently, substituted C 1 -C 6 alkyl. In certain embodiments, at least one Z is substituted methyl.
  • each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl. In certain embodiments, at least one substituent group is C 1 -C 6 alkoxy (e.g., at least one Z is C 1 -C 6 alkyl substituted with one or more C 1 -C 6 alkoxy). In another embodiment, each substituent group is, independently, C 1 -C 6 alkoxy (e.g., each Z is, independently, C 1 -C 6 alkyl substituted with one or more C 1 -C 6 alkoxy).
  • At least one C 1 -C 6 alkoxy substituent group is CH 3 O— (e.g., at least one Z is CH 3 OCH 2 -). In another embodiment, each C 1 -C 6 alkoxy substituent group is CH 3 O— (e.g., each Z is CH 3 OCH 2 -). In certain embodiments, at least one substituent group is halogen (e.g., at least one Z is C 1 -C 6 alkyl substituted with one or more halogen). In certain embodiments, each substituent group is, independently, halogen (e.g., each Z is, independently, C 1 -C 6 alkyl substituted with one or more halogen).
  • At least one halogen substituent group is fluoro (e.g., at least one Z is CH 2 FCH 2 -, CHF 2 CH 2 - or CF 3 CH 2 -).
  • each halo substituent group is fluoro (e.g., each Z is, independently, CH 2 FCH 2 -, CHF 2 CH 2 - or CF 3 CH 2 -).
  • at least one substituent group is hydroxyl (e.g., at least one Z is C1- C6 alkyl substituted with one or more hydroxyl).
  • each substituent group is, independently, hydroxyl (e.g., each Z is, independently, C 1 -C 6 alkyl substituted with one or more hydroxyl).
  • at least one Z is HOCH 2 -.
  • each Z is HOCH 2 -.
  • at least one Z is CH 3 -, CH 3 CH 2 -, CH 2 OCH 3 -, CH 2 F— or HOCH 2 -.
  • each Z is, independently, CH 3 -, CH 3 CH 2 -, CH 2 OCH 3 -, CH 2 F— or HOCH 2 -.
  • At least one Z group is C 1 -C 6 alkyl substituted with one or more Xx, aTQ ⁇ QUY QMOT Ib U]& UYPQ[QYPQY ⁇ Wc& C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z ⁇ 7B3 aTQ ⁇ QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY ⁇ Wc& ⁇ Z ⁇ 7 1 -C 6 alkyl, and X is O, S or NJ1.
  • At least one Z group is C 1 -C 6 alkyl substituted with one or more Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—) or azido.
  • each Z group is, independently, C 1 -C 6 alkyl substituted with one or XZ ⁇ Q Ib& aTQ ⁇ QUY QMOT Ib U] UYPQ[QYPQY ⁇ Wc C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z ⁇ 7B3 aTQ ⁇ QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY ⁇ Wc& ⁇ Z ⁇ 7 1 -C 6 alkyl, and X is O, S Docket No.: 121301-22520 ALN-511-WO or NJ1.
  • each Z group is, independently, C 1 -C 6 alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—) or azido.
  • Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—) or azido.
  • At least one Z group is —CH 2 Xx, wherein Xx is OJ1, NJ1J2, SJ1, B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z ⁇ 7B3 aTQ ⁇ QUY QMOT >*& >+ MYP >, U]& independently, H or C 1 -C 6 alkyl, and X is O, S or NJ1
  • at least one Z group is —CH 2 Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—) or azido.
  • each Z group is, independently, —CH 2 Xx, wherein each Xx is, UYPQ[QYPQY ⁇ Wc& C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z ⁇ 7B3 aTQ ⁇ QUY each J1, J2 and J3 is, independently, H or C 1 -C 6 alkyl, and X is O, S or NJ1.
  • each Z group is, independently, —CH 2 Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH 3 O—) or azido.
  • at least one Z is CH 3 -.
  • each Z is, CH 3 -.
  • the Z group of at least one monomer is in the (R)— configuration represented by the formula: or the formula: or the formula: IN certain embodiments, the Z group of each monomer of the formula is in the (R)— configuration.
  • the Z group of at least one monomer is in the (S)— configuration represented by the formula: or the formula: Docket No.: 121301-22520 ALN-511-WO or the formula:
  • the Z group of each monomer of the formula is in the (S)— configuration.
  • T3 is H or a hydroxyl protecting group.
  • T4 is H or a hydroxyl protecting group.
  • T3 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit.
  • T4 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit.
  • T3 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide.
  • T4 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide.
  • T3 is an internucleoside linking group attached to an oligomeric compound.
  • T4 is an internucleoside linking group attached to an oligomeric compound.
  • at least one of T3 and T4 comprises an internucleoside linking group selected from phosphodiester or phosphorothioate.
  • the dsRNA agent of the disclosure comprises a gapped motif.
  • the dsRNA agent of the disclosure OZX[ ⁇ U]Q] M ⁇ WQM] ⁇ ZYQ ⁇ QSUZY ZR R ⁇ ZX MNZ_ ⁇ 1 ⁇ Z MNZ_ ⁇ *- OZY ⁇ US_Z_] q'8'+o'PQZbc ⁇ UNZR_ ⁇ MYZ]cW nucleosides.
  • the dsRNA agent of the disclosure comprises at least one region ZR R ⁇ ZX MNZ_ ⁇ 2 ⁇ Z MNZ_ ⁇ *+ OZY ⁇ US_Z_] q'8'+o'PQZbc ⁇ UNZR_ ⁇ MYZ]cW Y_OWQZ]UPQ](
  • the dsRNA agent of the disclosure comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) comprises at least one (S)-cEt monomer of the formula: Docket No.: 121301-22520 ALN-511-WO , wherein Bx is heterocyclic base moiety.
  • monomers include sugar mimetics.
  • a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
  • a sugar mimetics include, but are not limited to, cyclohexenyl or morpholino.
  • Representative examples of a mimetic for a sugar-internucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances a mimetic is used in place of the nucleobase.
  • nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.
  • C. Intersugar Linkage Modifications Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming an oligomeric compound, e.g., an oligonucleotide. Such linking groups are also referred to as intersugar linkage.
  • linking groups are defined by the presence or absence of a phosphorus atom.
  • Representative non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane
  • Modified linkages can be used to alter, typically increase, nuclease resistance of the oligonucleotides.
  • linkages having a chiral atom can be prepared as racemic mixtures, as separate enantomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known to those skilled in the art.
  • the phosphate group in the linking group can be modified by replacing one of the oxygens with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown.
  • modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • one of the non-bridging phosphate oxygen atoms in the linkage can be replaced by any of the following: S, Se, BR 3 (R is hydrogen, alkyl, aryl), C (i.e.
  • the phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral; in other words a phosphorous atom in a phosphate group modified in this way is a stereogenic center.
  • the stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).
  • Phosphorodithioates have both non-bridging oxygens replaced by sulfur.
  • the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers.
  • modifications to both non-bridging oxygens, which eliminate the chiral center, e.g. phosphorodithioate formation can be desirable in that they cannot produce diastereomer mixtures.
  • the non-bridging oxygens can be independently any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).
  • the phosphate linker can also be modified by replacement of bridging oxygen, (i.e.
  • the replacement can occur at the either one of the linking oxygens or at both linking oxygens.
  • the bridging oxygen is the 3’-oxygen of a nucleoside, replacement with carbon is preferred.
  • the bridging oxygen is the 5’-oxygen of a nucleoside, replacement with nitrogen is preferred.
  • Modified phosphate linkages where at least one of the oxygen linked to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorous group are also referred to as “non-phosphodiester intersugar linkage” or “non-phosphodiester linker.”
  • the phosphate group can be replaced by non-phosphorus containing connectors, e.g. dephospho linkers.
  • Dephospho linkers are also referred to as non-phosphodiester linkers herein. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability.
  • Preferred embodiments include methylenemethylimino (MMI),methylenecarbonylamino, amides,carbamate and ethylene oxide linker.
  • a modification of a non-bridging oxygen can necessitate modification of 2’-OH, e.g., a modification that does not participate in cleavage of the neighboring intersugar linkage, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA.
  • Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Sp isomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Rp isomer, phosphorodithioates, phsophotriesters, aminoalkylphosphotrioesters, alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidate), and boranophosphonates.
  • phosphorodithioates e.g., methyl-phosphonate
  • selenophosphates e.g., N-alkyl
  • the dsRNA agent of the disclosure comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all) modified or nonphosphodiester linkages. In some embodiments, the dsRNA agent of the disclosure comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all) phosphorothioate linkages.
  • the dsRNA agent of the disclosures can also be constructed wherein the phosphate linker and the sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates.
  • a neutral surrogate backbone examples include the morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA) and backnone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates.
  • PNA peptide nucleic acid
  • aegPNA aminoethylglycyl PNA
  • bepPNA backnone-extended pyrrolidine PNA
  • the dsRNA agent of the disclosures described herein can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the dsRNA agent of the disclosures provided herein are all such possible isomers, as well as their racemic and optically pure forms. D. Terminal Modifications
  • the dsRNA agent further comprises a phosphate or phosphate mimic at the 5’-end of the antisense strand.
  • the phosphate mimic is a 5’-vinyl phosphonate (VP). Docket No.: 121301-22520 ALN-511-WO
  • the 5’-end of the antisense strand of the dsRNA agent does not contain a 5’-vinyl phosphonate (VP).
  • Ends of the iRNA agent of the disclosure can be modified. Such modifications can be at one end or both ends.
  • the 3 ⁇ and/or 5 ⁇ ends of an iRNA can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
  • the functional molecular entities can be attached to the sugar through a phosphate group and/or a linker.
  • the terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C- 3 ⁇ or C-5 ⁇ O, N, S or C group of the sugar.
  • the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs).
  • a linker/phosphate-functional molecular entity-linker/phosphate array is interposed between two strands of a double stranded oligomeric compound, this array can substitute for a hairpin loop in a hairpin-type oligomeric compound.
  • Terminal modifications useful for modulating activity include modification of the 5’ end of iRNAs with phosphate or phosphate analogs.
  • the 5’end of an iRNA is phosphorylated or includes a phosphoryl analog.
  • Exemplary 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Modifications at the 5’-terminal end can also be useful in stimulating or inhibiting the immune system of a subject.
  • the 5’-end of the oligomeric compound comprises the modification , wherein W, X and Y are each independently selected from the group consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR 3 (R is hydrogen, alkyl, aryl), BH 3 -, C (i.e.
  • a and Z are each independently for each occurrence absent, O, S, CH 2 , NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, wherein backbone of the alkylene can comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and/or at the end; and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A is replacing the oxygen linked to 5’ carbon of sugar.
  • W and Y together with the P to which they are attached can form an optionally substituted 5-8 membered heterocyclic, wherein W an Y are each independently O, S, NR’ or alkylene.
  • the heterocyclic is substituted with an aryl or heteroaryl.
  • one or both hydrogen on C5’ of the 5’- terminal nucleotides are replaced with a halogen, e.g., F.
  • Exemplary 5’-modifications include, but are not limited to, 5'-monophosphate ((HO) 2 (O)P-O- 5'); 5'-diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O- P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); 5'-alpha- thiotriphosphate; 5’-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidates ((HO) 2 (O)P-NH
  • exemplary 5’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO) 2 (X)P-O[-(CH 2 ) a -O-P(X)(OH)-O] b - 5', ((HO) 2 (X)P-O[-(CH 2 ) a -P(X)(OH)-O] b - 5', ((HO)2(X)P-[- (CH 2 ) a -O-P(X)(OH)-O] b - 5'; dialkyl terminal phosphates and phosphate mimics: HO[-(CH 2 ) a -O- P(X)(OH)-O] b - 5' , H 2 N[-(CH 2 ) a -O-P(X)(OH)-O] b - 5', H[-(CH 2 ) a -O-P(X)(OH)-O]
  • Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorescein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include targeting ligands. Terminal modifications can also be useful for cross-linking an oligonucleotide to another moiety; modifications useful for this include mitomycin C, psoralen, and derivatives thereof. E.
  • the compounds of the disclosure can be optimized for RNA interference by increasing the propensity of the iRNA duplex to disassociate or melt (decreasing the free energy of duplex association) by introducing a thermally destabilizing modification in the sense strand at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5’- end of the antisense strand, or at positions 2-9 of the 5’-end of the antisense strand).
  • This modification can increase the propensity of the duplex to disassociate or melt in the seed region of the antisense strand.
  • the thermally destabilizing modifications can include abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycerol nuceltic acid (GNA).
  • UUA unlocked nucleic acids
  • GAA glycerol nuceltic acid
  • acyclic nucleotide refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are 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’
  • ribose carbons or oxygen e.g., C1’, C2’, C3’, C4’ or O4’
  • nucleotide wherein B is a modified or unmodified nucleobase R 1 and R 2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • R 1 and R 2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).
  • the term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked "sugar” residue.
  • UNA also encompasses monomers with bonds between C1'-C4' being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons).
  • the C2'-C3' bond i.e. the covalent carbon-carbon bond between the C2' and C3' carbons
  • the acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings.
  • the acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.
  • the term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:
  • the thermally destabilizing modification 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.
  • Exemplary mismatch basepairs 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.
  • 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 Docket No.: 121301-22520 ALN-511-WO ribose sugars of the nucleotides.
  • the compounds of the disclosure such as siRNA or iRNA agent, contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand.
  • abasic nucleotide More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.
  • the thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.
  • Nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety.
  • Exemplary nucleobase modifications are: .
  • the 2’-5’ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5’ end of the sense strand to avoid sense strand activation by RISC.
  • compounds of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2’-H, 2’-OH and 2’-OMe).
  • these L sugar modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5’ end of the sense strand to avoid sense strand activation by RISC.
  • the iRNA agent of the disclosure is conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, Docket No.: 121301-22520 ALN-511-WO [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,
  • At least one strand of the iRNA agent of the disclosure disclosed herein is 5’ phosphorylated or includes a phosphoryl analog at the 5’ prime terminus.
  • 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5'-monophosphate ((HO) 2 (O)P-O-5'); 5'-diphosphate ((HO) 2 (O)P-O- P(HO)(O)-O-5'); 5'-triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7- methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O- (HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)P-O-5'); 5
  • RNAi agents of the Disclosure Comprising Motifs
  • the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in U.S. Patent Nos.9,796,974 and 10,668,170, and U.S.
  • Patent Publication Nos.2014/288158, 2018/008724, 2019/038768, and 2020/353097 the entire contents of each of which are incorporated herein by reference.
  • one or more motifs of three identical modifications on three consecutive nucleotides may be introduced into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site.
  • the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand.
  • the RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand.
  • the iRNA agent of the disclosure is a double ended bluntmer of 19 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 7,8,9 from the 5’end.
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.
  • the iRNA agent of the disclosure is a double ended bluntmer of 20 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three Docket No.: 121301-22520 ALN-511-WO consecutive nucleotides at positions 8,9,10 from the 5’end.
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.
  • the iRNA agent of the disclosure is a double ended bluntmer of 21 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end.
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.
  • the iRNA agent of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end; the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end, wherein one end of the iRNA is blunt, while the other end is comprises a 2 nt overhang.
  • the 2 nt overhang is at the 3’-end of the antisense.
  • the iRNA agent further comprises a ligand (e.g., GalNAc 3 ).
  • the iRNA agent of the disclosure comprises a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1- 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with sense strand, thereby forming a 3' single
  • the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.
  • the iRNA agent of the disclosure comprises a sense and antisense strands, wherein said iRNA agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at position 11,12,13 from the 5’ end; wherein said 3’ end of said first strand and said 5’ end of said second strand form a blunt end and said second strand is 1-4 nucleotides longer at its 3’ end than the first strand, wherein the Docket No.: 121301-22520 ALN-511-WO duplex region region which is at least 25 nucleotides in length, and said second strand is sufficiently complemenatary to a target mRNA along
  • the iRNA agent further comprises a ligand (e.g., GalNAc 3 ).
  • the sense strand of the iRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.
  • the sense strand can contain at least one motif of three 2’-F modifications on three consecutive nucleotides within 7-15 positions from the 5’end.
  • the antisense strand of the iRNA agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.
  • the antisense strand can contain at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides within 9-15 positions from the 5’end.
  • the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5’-end.
  • the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1 st nucleotide from the 5’-end of the antisense strand, or, the count starting from the 1 st paired nucleotide within the duplex region from the 5’- end of the antisense strand.
  • the cleavage site in the antisense strand may also change according to the length of the duplex region of the iRNA from the 5’-end.
  • the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide.
  • the antisense strand also contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand.
  • the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand.
  • the antisense strand also contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.
  • the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end, and wherein the Docket No.: 121301-22520 ALN-511-WO antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.
  • the iRNA agent of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch can occur in the overhang region or the duplex region.
  • the base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used).
  • A:U is preferred over G:C
  • G:U is preferred over G:C
  • Mismatches e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.
  • the iRNA agent of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5’- end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex.
  • the nucleotide at the 1 position within the duplex region from the 5’-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT.
  • At least one of the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair.
  • the first base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair.
  • the nucleotide at the 3’-end of the sense strand is deoxythimidine (dT).
  • the nucleotide at the 3’-end of the antisense strand is deoxythimidine (dT).
  • there is a short sequence of deoxythimidine nucleotides for example, two dT nucleotides on the 3’-end of the sense or antisense strand.
  • compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein.
  • a vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure.
  • a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5’ end of the antisense strand of the dsRNA.
  • Vinyl phosphonate modifications are also contemplated for the compositions and methods of the instant disclosure.
  • the disclosure relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene having reduced off-target effects as described in U.S. Patent Nos. 10,233448, 10,612,024, and 10,612,027, and U.S. Patent Publication Nos.2017/275626, 2019/241891, 2019/241893, and 2021/017519, the entire contents of each of which are incorporated herein by reference.
  • dsRNA double-stranded RNA
  • a motif comprising, e.g., a thermally destabilizing nucleotide, e.g., i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and/or iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) may be introduced into the sense strand.
  • the dsRNA agent of the disclosure does not contain any 2’-F modification.
  • the dsRNA agent of the disclosure only contains 2’-F and 2’-OMe modifications.
  • the sense strand and/or antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages.
  • the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages.
  • the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages.
  • the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.
  • each of the sense and antisense strands of the dsRNA agent has 15-30 nucleotides.
  • the sense strand has 19-22 nucleotides, and the antisense strand has 19- 25 nucleotides.
  • the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides.
  • the nucleotide at position 1 of the 5’-end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, U, and dT.
  • at least one of the first, second, and third base pair from the 5’-end of the antisense strand is an AU base pair.
  • the antisense strand of the dsRNA agent of the disclosure is 100% complementary to a target RNA to hybridize thereto and inhibits its expression through RNA interference.
  • the antisense strand of the dsRNA agent of the disclosure is at Docket No.: 121301-22520 ALN-511-WO least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to a target RNA.
  • the disclosure relates to a dsRNA agent as defined herein capable of inhibiting the expression of a target gene.
  • the dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides.
  • the sense strand contains at least one thermally destabilizing nucleotide, wherein at least one of said thermally destabilizing nucleotide occurs at or near the site that is opposite to the seed region of the antisense strand (i.e. at position 2-8 of the 5’-end of the antisense strand, or at positions 2-9 of the 5’-end of the antisense strand).
  • Each of the embodiments and aspects described in this specification relating to the dsRNA represented by formula (I) can also apply to the dsRNA containing the thermally destabilizing nucleotide.
  • the thermally destabilizing nucleotide can occur, for example, between positions 14-17 of the 5’-end of the sense strand when the sense strand is 21 nucleotides in length.
  • the antisense strand contains at least two modified nucleic acids that are smaller than a sterically demanding 2’-OMe modification.
  • the two modified nucleic acids that are smaller than a sterically demanding 2’-OMe are separated by 11 nucleotides in length.
  • the two modified nucleic acids are at positions 2 and 14 of the 5’end of the antisense strand.
  • the dsRNA agent further comprises at least one ASGPR ligand.
  • the ASGPR ligand is one or more GalNAc derivatives attached a bivalent or trivalent branched linker, such as: one example, the ASGPR ligand is attached to the 3’ end of the sense strand.
  • the dsRNA agent as defined herein can comprise i) a phosphorus-containing group at the 5’-end of the sense strand or antisense strand; ii) with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’- end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand); and iii) a ligand, such as a ASGPR ligand (e.g., one or more GalNAc derivatives
  • the ligand may be at the 3’-end of the sense strand.
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; and (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2’- OMe 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’-OMe modifications at positions 1, 3, Docket No.: 121301-22520 ALN-511-WO 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2’F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2’- OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5’ end); and (iv) 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’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’F modifications at positions 2,
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2’-F modifications at positions 7, and 9, and a desoxy-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’-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2’-F 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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2’-F modifications at positions 7, 9, 11, 13, and 15; and (iv) 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’-OMe modifications at Docket No.: 121301-22520 ALN-511-WO positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 9, and 12 to 21, and 2’-F modifications at positions 10, and 11; and (iv) 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’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5’ end); and (i) a sense
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2’-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and (iv) 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’-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 5, Docket No.: 121301-22520 ALN-511-WO 7, 8, 10 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 9,
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5’ end); and (iii
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 19 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2’-F modifications at positions 5, and 7 to 9; and (iv) 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 21 nucleotides; (ii) 2’-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2’-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5’ end); and (iii
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 18-23 nucleotides; (ii) three consecutive 2’-F modifications at positions 7-15; and (b) an antisense strand having: (i) a length of 18-23 nucleotides; (ii) at least 2’-F modifications anywhere on the strand; and (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides Docket No.: 121301-22520 ALN-511-WO overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex.
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 18-23 nucleotides; (ii) less than four 2’-F modifications; (b) an antisense strand having: (i) a length of 18-23 nucleotides; (ii) less than twelve 2’-F modfication; and (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex.
  • the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 19-35 nucleotides; (ii) less than four 2’-F modifications; (b) an antisense strand having: (i) a length of 19-35 nucleotides; (ii) less than twelve 2’-F modfication; and (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); and wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex.
  • the dsRNA agents of the present disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) a liophilic moiety (e.g., a C 22 hydrocarbon chain); (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 4, 6, 8 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 14, and 16, a 2’-deoxy-modified nucleotide at position 5, and a GNA at position 7 (counting from the 5’ end); and (iii
  • the dsRNA agents of the present disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) a liophilic moiety (e.g., a C 22 hydrocarbon chain); (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 5, 7 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 14, and 16 (counting from the 5’ end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2,
  • the dsRNA agents of the present disclosure comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have less than 20% , less than 15% and less than 10% non-natural nucleotide.
  • non-natural nucleotide includes acyclic nucleotides, LNA, HNA, CeNA, 2’- methoxyethyl, 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), a 2'- O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F, and others.
  • the dsRNA agents of the present disclosure comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have greater than 80% , greater than 85% and greater than 90% natural nucleotide, such as 2’-OH, 2’-deoxy and 2’-OMe are natural nucleotides.
  • the dsRNA agents of the present disclosure comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have 100% natural nucleotide, such as 2’-OH, 2’-deoxy and 2’-OMe are natural nucleotides.
  • RNA agent of the disclosure includes WO2007/091269, US Patent No.7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520, which are hereby incorporated by reference in their entirety.
  • 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the iRNA agent of the disclosure is modified.
  • each of the sense and antisense strands of the iRNA agent is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2’-methoxyethyl, 2’- O-methyl, 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), a 2'-O- dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.
  • acyclic nucleotides LNA, HNA, CeNA, 2’-methoxyethyl, 2’- O-methyl, 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), a 2'
  • each of the sense and antisense strands of the iRNA agent contains at least two different modifications. Docket No.: 121301-22520 ALN-511-WO
  • the dsRNA agent of the disclosure contains one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 2’-F modification(s). In one example, dsRNA agent of the disclosure contains nine or ten 2’-F modifications.
  • the iRNA agent of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.
  • the phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.
  • the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern.
  • the alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.
  • the iRNA comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.
  • the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.
  • Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide.
  • the sense strand and/or antisense strand of the iRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages.
  • the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages.
  • the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages.
  • the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.
  • the disclosure relates to a iRNA agent capable of inhibiting the expression of a target gene.
  • the iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides.
  • the sense strand contains at least one thermally destabilizing nucleotide, wherein at at least one said thermally destabilizing nucleotide occurs at or near the site that is opposite to the seed region of the antisense strand (i.e .at position 2-8 of the 5’-end of the antisense strand, or at Docket No.: 121301-22520 ALN-511-WO positions 2-9 of the 5’-end of the antisense strand).
  • the thermally destabilizing nucleotide occurs between positions 14-17 of the 5’-end of the sense strand when the sense strand is 21 nucleotides in length.
  • the antisense strand contains at least two modified nucleic acids that are smaller than a sterically demanding 2’-OMe modification.
  • the two modified nucleic acids that is smaller than a sterically demanding 2’-OMe are separated by 11 nucleotides in length.
  • the two modified nucleic acids are at positions 2 and 14 of the 5’end of the antisense strand.
  • the compound of the disclosure disclosed herein is a miRNA mimic.
  • miRNA mimics are double stranded molecules (e.g., with a duplex region of between about 16 and about 31 nucleotides in length) and contain one or more sequences that have identity with the mature strand of a given miRNA.
  • a miRNA mimic has designs similar to as described above for double-stranded iRNAs.
  • a miRNA mimic comprises a duplex region of between 16 and 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide), and all of the Cs and Us; the antisense strand modifications can comprise 2' F modification of all of the Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with a 2 nucleotide 3 ' overhang. V.
  • a lipohilic moiety of the disclosure can comprise at least one C10-C26 hydrocarbon chain.
  • a C 22 hydrocarbon chain e.g., saturated or unsaturated
  • octanol-water partition coefficient logK ow
  • K ow 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 laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci.41:1407-21 (2001), which is incorporated herein by reference in its entirety).
  • a chemical substance is lipophilic in character when its logK ow exceeds 0.
  • the lipophilic moiety possesses a logK ow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10.
  • the logK ow of 6- amino hexanol for instance, is predicted to be approximately 0.7.
  • the logK ow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.
  • 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 C 22 hydrocarbon chain can increase or decrease the partition coefficient (e.g., logK ow ) value of the C 22 hydrocarbon chain.
  • the hydrophobicity of the dsRNA agent, conjugated to one or more C 22 hydrocarbon chains can be measured by its protein binding characteristics. For instance, 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 mobility shift assay (EMSA) using human serum albumin protein.
  • ESA electrophoretic mobility shift assay
  • the hydrophobicity of the dsRNA agent measured by fraction of unbound dsRNA 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 siRNA.
  • the one or more C 22 hydrocarbon chains is an aliphatic, alicyclic, or polyalicyclic compound is an aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compound.
  • the hydrocarbon chain may comprise various substituents and/or one or more heteroatoms, such as an oxygen or nitrogen atom.
  • the one or more C 22 hydrocarbon chains may be attached to the iRNA agent by any method known in the art, including via a functional grouping already present in the lipophilic moiety or introduced into the iRNA agent, such as a hydroxy group (e.g., —CO—CH 2 —OH).
  • the functional groups already present in the C 22 hydrocarbon chain 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 C 22 hydrocarbon chain 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 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.
  • the C 22 hydrocarbon chain is conjugated to the dsRNA agent via a linker 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 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 one or more 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 one or more C22 hydrocarbon chains is a C22 alcohol, e.g. the C22 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, and cis-4,7,10,13,16,19-docosahexanol.
  • the one or more C 22 hydrocarbon chains is not cis-4,7,10,13,16,19- docosahexanoic acid. In one embodiment, the one or more C 22 hydrocarbon chains is not cis- 4,7,10,13,16,19-docosahexanol. In one embodiment, the one or more C 22 hydrocarbon chains is not cis-4,7,10,13,16,19-docosahexanoic acid and is not cis-4,7,10,13,16,19-docosahexanol.
  • the one or more C 22 hydrocarbon chains is a C 22 amide, e.g., the C 22 amide is selected 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 C 22 amide is selected from the group consisting of (E)-Docos-4-enamide, (E)-Docos-5-enamide, (Z)- Docos-9-enamide, (E)-Do
  • more than one C 22 hydrocarbon chains can be incorporated into the double-strand iRNA agent, particularly when the C 22 hydrocarbon chains has a low lipophilicity or hydrophobicity.
  • two or more C 22 hydrocarbon chains are incorporated into the same strand of the double-strand iRNA agent.
  • each strand of the double-strand iRNA agent has one or more C 22 hydrocarbon chains incorporated.
  • two or more C 22 hydrocarbon chains are incorporated into the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the double-stranded iRNA agent.
  • the one or more C 22 hydrocarbon chains may be conjugated to the iRNA agent via a direct attachment to the ribosugar of the iRNA agent.
  • the one or more C 22 hydrocarbon chains may be conjugated to the double-strand iRNA agent via a linker or a carrier.
  • the one or more C 22 hydrocarbon chains may be conjugated to the iRNA agent via one or more linkers (tethers).
  • the one or more C 22 hydrocarbon chains is conjugated to the 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 lipophilic moiety (e.g., C 22 hydrocarbon chain) is attached at the 2’ position on an internal nucleotide of the sense or antisense strand. In one embodiment, the lipophilic moiety (e.g., C 22 hydrocarbon chain) is attached at the 2’ position on the nucleotide at any of positions 4-8 and 13-18 on the sense strand (e.g., position 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, 18) counting from the 5’ end. In one embodiment, the lipophilic moiety (e.g., C 22 hydrocarbon chain) is attached at the 2’ position on the nucleotide at position 6 or 16 on the sense strand counting from the 5’ end. A.
  • Linkers/Tethers Docket No.: 121301-22520 ALN-511-WO Linkers/Tethers are connected to the one or more C 22 hydrocarbon chains at a “tethering attachment point (TAP).”
  • Linkers/Tethers may include any C 1 -C 100 carbon-containing moiety, (e.g. C 1 -C 75 , C 1 -C 50 , C 1 -C 20 , C 1 -C 10 ; C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , or C 10 ), and may have at least one nitrogen atom.
  • 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.
  • linkers/tethers underlined
  • linkers/tethers include TAP-(CH 2 ) n NH-; TAP- C(O)(CH 2 ) n NH-; TAP-NR’’’’(CH 2 ) n NH-, TAP-C(O)-(CH 2 ) n -C(O)-; TAP-C(O)-(CH 2 ) n -C(O)O-; TAP- C(O)-O-; TAP-C(O)-(CH 2 ) n -NH-C(O)-; TAP-C(O)-(CH 2 ) n -; TAP-C(O)-NH-; TAP-C(O)-; TAP-C(O)-; TAP- (CH 2
  • n is 5, 6, or 11.
  • the nitrogen may form part of a terminal oxyamino group, e.g., -ONH 2 , or hydrazino group, -NHNH 2 .
  • the linker/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.
  • Preferred tethered ligands may include, e.g., TAP- (CH 2 ) n NH(LIGAND); TAP-C(O)(CH 2 ) n NH(LIGAND); TAP-NR’’’’(CH 2 ) n NH(LIGAND); TAP- (CH 2 ) n ONH(LIGAND); TAP-C(O)(CH 2 ) n ONH(LIGAND); TAP-NR’’’’(CH 2 ) n ONH(LIGAND); TAP-(CH 2 ) n NHNH 2 (LIGAND), TAP-C(O)(CH 2 ) n NHNH 2 (LIGAND); TAP- NR’’’’(CH 2 ) n NHNH 2 (LIGAND); TAP-C(O)-(CH 2 ) n -C(O)(LIGAND); TAP-C(O)-(CH 2 ) n - C(O)
  • amino terminated linkers/tethers e.g., NH 2 , ONH 2 , NH 2 NH 2
  • the 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.
  • electrophilic moieties include, e.g., an aldehyde, 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- (CH 2 ) n CHO; TAP-C(O)(CH 2 ) n CHO; or TAP-NR’’’’(CH 2 ) n CHO, in which n is 1-6 and R’’’’ is C 1 -C 6 alkyl; or TAP-(CH 2 ) n C(O)ONHS; TAP-C(O)(CH 2 ) n C(O)ONHS; or TAP-NR’’’’(CH 2 ) n C(O)ONHS, in which n is 1-6 and R’’’’’ is C 1 -C 6 alkyl; TAP-(CH 2 ) n C(O)OC 6 F 5 ; TAP-C(O)(CH 2 ) n C(O) OC 6 F 5 ; or TAP-NR’’’’(CH 2 ) n C(O) OC 6 F 5 , in which n is 1-11 and R’
  • 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 e.g., a thiol or amino group
  • the monomer can include a phthalimido group (K) at the terminal position of the
  • other protected amino groups can be at the terminal position of the 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).
  • B. Cleavable 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).
  • at least one of the linkers/tethers can be an esterase cleavable linker (e.g., an ester group).
  • at least one of the linkers/tethers can be a phosphatase cleavable linker (e.g., a phosphate group).
  • 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 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 human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some tethers will have a linkage group that is cleaved at a preferred pH, thereby Docket No.: 121301-22520 ALN-511-WO 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 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.
  • an iRNA agent that targets an mRNA in liver cells can be conjugated to a tether that includes an ester group. Liver cells are rich in esterases, and therefore the tether will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Cleavage of the tether releases the iRNA agent from a ligand that is attached to the distal end of the tether, thereby potentially enhancing silencing activity of the iRNA agent.
  • Tethers that contain peptide bonds can be conjugated to iRNA agents target to cell types rich in peptidases, such as liver cells and synoviocytes.
  • iRNA agent targeted to synoviocytes such as for the treatment of an inflammatory disease (e.g., rheumatoid arthritis)
  • 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.
  • 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.
  • 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.
  • 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).
  • C. Redox Cleavable Linking Groups 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 Docket No.: 121301-22520 ALN-511-WO moiety and particular targeting agent
  • 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 most 10% in the blood.
  • useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).
  • the rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
  • D. Phosphate-Based Cleavable Linking Groups are cleaved by agents that degrade or hydrolyze the phosphate group.
  • An example of an agent that cleaves phosphate groups in cells 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 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 OWQM ⁇ MNWQ S ⁇ Z_[] OMY TM ⁇ Q ⁇ TQ SQYQ ⁇ MW RZ ⁇ X_WM g7nBBg& 7#C$C& Z ⁇ gC7#C$( 5 [ ⁇ QRQ ⁇ QP embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl.
  • Ester-based 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 — C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.
  • G. Peptide-Based Cleaving Groups 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 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.
  • the linkers can also includes biocleavable linkers that are nucleotide and non-nucleotide linkers or combinations thereof that connect two parts of a molecule, for example, one or both strands of two individual siRNA molecules to generate a bis(siRNA). In some embodiments, mere electrostatic or stacking interaction between two individual siRNAs can represent a linker.
  • the non- nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and derivatives thereof, aliphatic, alicyclic, hetercyclic, and combinations thereof.
  • at least one of the linkers (tethers) 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: Docket No.: 121301-22520 ALN-511-WO Docket No.: 121301-22520 ALN-511-WO
  • the one or more C 22 hydrocarbon chains is conjugated to the iRNA 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 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.
  • the carrier is a cyclic group having an amine
  • the carrier may be Docket No.: 121301-22520 ALN-511-WO pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.
  • a ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is 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 C 22 hydrocarbon chains can be directly attached to the carrier or attached to the carrier an 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 present in an iRNA agent. a.
  • Cyclic sugar replacement-based monomers e.g., sugar replacement-based ligand-conjugated monomers
  • RRMS monomer compounds are also referred to herein as RRMS monomer compounds.
  • the carriers may have the 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 CH2.
  • the carriers are described below as an entity, which can be incorporated into a strand.
  • the structures also encompass the situations 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 -CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.
  • 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 ;
  • R 5 , R 6 , R 11 , and R 12 is, independently, a ligand, H, C 1 -C 6 alkyl optionally substituted 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
  • R b is P(O)(O-)H, P(OR 15 )N(R 16 ) 2 or L-R 17 ;
  • 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- 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). Docket No.: 121301-22520 ALN-511-WO .
  • 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 (- CH 2 OFG 1 in D).
  • OFG 2 is preferably attached directly to one of the carbons in the five-membered ring (-OFG 2 in D).
  • -CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or -CH 2 OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4.
  • CH 2 OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.
  • -CH 2 OFG 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, e.g. restriction resulting from the presence of a ring.
  • CH 2 OFG 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. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH 2 OFG 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: .
  • the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR 11 R 12 . .
  • OFG 2 is preferably attached directly to one of the carbons in the six- membered ring (-OFG 2 in E).
  • -(CH 2 ) n OFG 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.
  • - (CH 2 ) n OFG 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., -(CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; -(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; - (CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH 2 ) n OFG 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 linkage, e.g. restriction resulting from the presence of a ring.
  • linkages e.g., carbon-carbon bonds
  • -(CH 2 ) n OFG 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 tethering attachment point is preferably nitrogen.
  • the carrier may be based on the piperazine ring system (F), e.g., X is (G), e.g., X is N(CO)R 7
  • 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 (-CH2OFG 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 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 Docket No.: 121301-22520 ALN-511-WO 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 Docket No.: 121301-22520 ALN-511-WO bonds
  • bond rotation e.g. restriction resulting from the presence of a ring.
  • CH 2 OFG 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. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH 2 OFG 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).
  • R’’’ can be, e.g., C 1 - C 6 alkyl, preferably CH 3 .
  • 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).
  • -(CH 2 ) n OFG 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.
  • -(CH 2 ) n OFG 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., -(CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; - (CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; -(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-3; -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-5; or - (CH 2 ) n OFG 1 may be attached to C-5 and OFG 2 may be attached to C-4.
  • the decalin or indane-based monomers may 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
  • - (CH 2 ) n OFG 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 centers bearing CH 2 OFG 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 substituents at C-1 and C-6 are trans with respect to one another.
  • the tethering attachment point is preferably C-6 or C-7.
  • Docket No.: 121301-22520 ALN-511-WO Other carriers may include those based on 3-hydroxyproline (J). .
  • -(CH2)nOFG 1 and OFG 2 may be cis or trans with respect to one another. 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. 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 U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties. b.
  • Acyclic sugar replacement-based monomers e.g., sugar replacement-based ligand-conjugated monomers
  • RRMS ribose replacement monomer subunit
  • Preferred 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.
  • the tertiary carbon can have either the R or S 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 entireties.
  • the one or more C 22 hydrocarbon chains is conjugated to one or more internal positions on at least one 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 position 1 counting from the 5’ end).
  • the one or more C 22 hydrocarbon chains is 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 Docket No.: 121301-22520 ALN-511-WO and positions 1 and 2 counting from the 5’ end).
  • the one or more C 22 hydrocarbon chains 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 one or more C 22 hydrocarbon chains is conjugated to one or more internal positions on at least one strand, except the cleavage site region of the sense strand, for instance, the one or more C 22 hydrocarbon chains is not conjugated to positions 9-12 counting from the 5’-end of the sense strand, for example, the one or more C 22 hydrocarbon chains 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 strand.
  • the one or more C 22 hydrocarbon chains is conjugated to one or more internal positions on at least one strand, which exclude the cleavage site region of the antisense strand.
  • the internal positions exclude positions 12-14 counting from the 5’-end of the antisense strand.
  • the one or more C 22 hydrocarbon chains is 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 one or more C 22 hydrocarbon chains is conjugated to 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 one or more C 22 hydrocarbon chains is conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’end of each strand. In one embodiment, the one or more C 22 hydrocarbon chains is conjugated to position 6 on the sense strand, counting from the 5’end of each strand.
  • the one or more C 22 hydrocarbon chains is conjugated to position 16 on the sense strand, counting from the 5’end of each strand. In some embodiments, the one or more C 22 hydrocarbon chains is conjugated to a nucleobase, sugar moiety, or internucleosidic phosphate linkage of the dsRNA agent.
  • siRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.
  • exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.
  • An siRNA can be made by separately synthesizing a single stranded RNA molecule, or each respective strand of a double-stranded RNA molecule, after which the component strands can then be annealed.
  • a large bioreactor e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given siRNA.
  • the OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide.
  • ribonucleotides amidites are used.
  • Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the siRNA.
  • the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.
  • Organic synthesis can be used to produce a discrete siRNA species.
  • the complementary of the species to a particular target gene can be precisely specified.
  • the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.
  • B. dsiRNA Cleavage siRNAs can also be made by cleaving a larger siRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used: 1. In vitro transcription.
  • dsiRNA is produced by transcribing a nucleic acid (DNA) segment in both directions.
  • DNA nucleic acid
  • the HiScribeTM RNAi transcription kit provides a vector and a method for producing a dsiRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsiRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsiRNA is produced.
  • RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes.
  • dsRNA is cleaved in vitro into siRNAs, for example, using a Dicer or comparable RNAse III- based activity.
  • the dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g., a purified RNAse or RISC complex (RNA-induced silencing complex ).
  • purified components e.g., a purified RNAse or RISC complex (RNA-induced silencing complex ).
  • RISC complex RNA-induced silencing complex
  • dsiRNA cleavage generally produces a plurality of siRNA species, each being a particular 21 to 23 nt fragment of a source dsiRNA molecule.
  • siRNAs that include sequences Docket No.: 121301-22520 ALN-511-WO complementary to overlapping regions and adjacent regions of a source dsiRNA molecule may be present.
  • the siRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation.
  • the siRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried siRNA can then be resuspended in a solution appropriate for the intended formulation process.
  • the one or more C 22 hydrocarbon chains is conjugated to the dsRNA agent via a nucleobase, sugar moiety, or internucleosidic linkage.
  • Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms.
  • the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a C 22 hydrocarbon chain.
  • Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position.
  • the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a C 22 hydrocarbon chain.
  • the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing.
  • the one or more C 22 hydrocarbon chains may be conjugated to a nucleobase via a linker containing an alkyl, alkenyl or amide linkage. Conjugation to sugar moieties of nucleosides can occur at any carbon atom.
  • Exemplary carbon atoms of a sugar moiety that the one or more C 22 hydrocarbon chains can be attached to include the 2', 3', and 5' carbon atoms.
  • the one or more C 22 hydrocarbon chains can also be attached to the 1' position, such as in an abasic residue.
  • the the one or more C 22 hydrocarbon chains may be conjugated to a sugar moiety, via a 2’-O modification, with or without a linker. Internucleosidic linkages can also bear the one or more C 22 hydrocarbon chains.
  • the the one or more C 22 hydrocarbon chains can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom.
  • the one or more C 22 hydrocarbon chains can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
  • an oligonucleotide is attached to a conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, and the like) on the oligonucleotide with a reactive group on the conjugate moiety.
  • a reactive group e.g., OH, SH, amine, carboxyl, aldehyde, and the like
  • one reactive group is electrophilic and the other is nucleophilic.
  • an electrophilic group can be a carbonyl-containing functionality and a nucleophilic group can be an amine or thiol.
  • a first (complementary) RNA strand and a second (sense) RNA strand can be synthesized separately, wherein one of the RNA strands comprises a pendant C 22 hydrocarbon chain, and the first and second RNA strands can be mixed to form a dsRNA.
  • the step of synthesizing the RNA strand preferably involves solid-phase synthesis, wherein individual nucleotides are joined QYP ⁇ Z QYP ⁇ T ⁇ Z_ST ⁇ TQ RZ ⁇ XM ⁇ UZY ZR UY ⁇ Q ⁇ Y_OWQZ ⁇ UPQ ,o'.o [TZ][TZPUQ] ⁇ Q ⁇ NZYP] UY OZY]QO_ ⁇ U ⁇ Q synthesis cycles.
  • the C 22 hydrocarbon chain having a phosphoramidite group is coupled to ⁇ TQ ,j'QYP Z ⁇ .o'QYP ZR QU ⁇ TQ ⁇ ⁇ TQ RU ⁇ ] ⁇ #OZX[WQXQY ⁇ M ⁇ c$ Z ⁇ ]QOZYP #]QY]Q$ EB5 ] ⁇ MYP UY ⁇ TQ WM] ⁇ synthesis cycle.
  • the nucleotides are initially in the form of nucleoside phosphoramidites.
  • a further nucleoside phosphoramidite is linked to the -OH group of the previously incorporated nucleotide.
  • the oligonucleotides can be synthesized using protocols known in the art, for example, as described in Caruthers et al., Methods in Enzymology (1992) 211:3-19; WO 99/54459; Wincott et al., Nucl. Acids Res. (1995) 23:2677-2684; Wincott et al., Methods Mol. Bio., (1997) 74:59; Brennan et al., Biotechnol. Bioeng. (1998) 61:33-45; and U.S. Pat. No.6,001,311; each of which is hereby incorporated by reference in its entirety.
  • syntheses can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.), or by methods such as those described in Usman et al., J. Am. Chem. Soc. (1987) 109:7845; Scaringe, et al., Nucl. Acids Res. (1990) 18:5433; Wincott, et al., Nucl. Acids Res. (1990) 23:2677-2684; and Wincott, et al., Methods Mol. Bio.
  • nucleic acid molecules of the present disclosure may be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., Science (1992) 256:9923; WO 93/23569; Shabarova et al., Nucl. Acids Res. (1991) 19:4247; Bellon et al., Nucleosides & Nucleotides (1997) 16:951; Bellon et al., Bioconjugate Chem. (1997) 8:204; or by hybridization following synthesis and/or deprotection.
  • the nucleic acid molecules can be purified by gel Docket No.: 121301-22520 ALN-511-WO electrophoresis using conventional methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water.
  • the dsRNA agent of the disclosure is further modified by covalent attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached dsRNA agent of the disclosure including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound.
  • a preferred list of conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • the dsRNA agent further comprises a targeting ligand that targets a receptor which mediates delivery to a specific tissue, e.g., liver tissue.
  • a targeting ligand that targets a receptor which mediates delivery to a specific tissue, e.g., liver tissue.
  • These targeting ligands can be conjugated in combination with the one or more C 22 hydrocarbon chains to enable specific systemic delivery.
  • a targeting ligand e.g., one or more GalNAc derivatives
  • a targeting ligand e.g., one or more GalNAc derivatives
  • a targeting ligand e.g., one or more GalNAc derivatives, is not conjugated to a dsRNA agent of the disclosure in combination with the one or more C 22 hydrocarbon chains.
  • Exemplary targeting ligands that targets the receptor mediated delivery to an adipose tissue are peptide ligands such as Angiopep-2, lipoprotein receptor related protein (LRP) ligand, bEnd.3 cell binding ligand; transferrin receptor (TfR) ligand (which can utilize iron transport system in brain and cargo transport into the brain parenchyma); manose receptor ligand (which targets olfactory ensheathing cells, glial cells), glucose transporter protein, and LDL receptor ligand.
  • Conjugate groups amenable to the present disclosure include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • cholic acid Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053
  • a thioether e.g., hexyl-S-tritylthiol
  • a thiocholesterol (Oberhauser et al., Nucl.
  • Ligands can include naturally occurring molecules, or recombinant or synthetic molecules.
  • Exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide- co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG- 5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG] 2 , polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptido
  • psoralen mitomycin C
  • porphyrins e.g., TPPC4, texaphyrin, Sapphyrin
  • polycyclic aromatic hydrocarbons e.g., phenazine, dihydrophenazine
  • artificial endonucleases e.g., EDTA
  • lipophilic molecules e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimeth
  • biotin e.g., biotin
  • transport/absorption facilitators e.g., naproxen, aspirin, vitamin E, folic acid
  • synthetic ribonucleases e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin OZRMO ⁇ Z ⁇ ]& WU[Z[ZWc]MOOTM ⁇ UPQ& MY MO ⁇ U ⁇ M ⁇ Z ⁇ ZR [,1 A5D VUYM]Q& MY MO ⁇ U ⁇ M ⁇ Z ⁇
  • Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., 8 Z ⁇ @ [Q[ ⁇ UPQ]3 l& q& Z ⁇ r [Q[ ⁇ UPQ]3 B'XQ ⁇ TcW [Q[ ⁇ UPQ]3 MdM[Q[ ⁇ UPQ]3 [Q[ ⁇ UPQ] TM ⁇ UYS one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
  • a peptidomimetic (also referred to herein as an Docket No.: 121301-22520 ALN-511-WO oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide.
  • the peptide or peptidomimetic ligand can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • Exemplary amphipathic peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S.
  • endosomolytic ligand refers to molecules having endosomolytic properties.
  • Endosomolytic ligands promote the lysis of and/or transport of the composition of the disclosure, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell.
  • Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and brached polyamines, e.g.
  • spermine cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.
  • Exemplary endosomolytic/fusogenic peptides include, but are not limited to, GALA; EALA; INF-7; Inf HA-2; diINF-7; diINF-3; GLF; GALA-INF3; INF-5, n is norleucine; JTS-1; ppTG1; ppTG20; KALA; HA; Melittin; H 5 WYG; and CHK 6 HC.
  • fusogenic lipids fuse with and consequently destabilize a membrane. Fusogenic lipids usually have small head groups and unsaturated acyl chains.
  • Exemplary fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3- phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k- DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also refered to as XTC herein).
  • DOPE 1,2-dileoyl-sn-3-
  • Exemplary cell permeation peptides include, but are not limited to, penetratin; Tat fragment 48-60; signal sequence based peptide; PVEC; transportan; amphiphilic model peptide; Arg9; 6MO ⁇ Q ⁇ UMW OQWW aMWW [Q ⁇ XQM ⁇ UYS [Q[ ⁇ UPQ3 @@',03 OQO ⁇ Z[UY D*3 l'PQRQY]UY3 q'PQRQY]UY3 DE',23 indolicidin; RFGF; RFGF analogue; and bactenecin.
  • NH 2 alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid
  • NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).
  • targeting ligand refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment.
  • Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.
  • Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc 2 and GalNAc 3 (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); D- mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, Glucose, multivalent Glucose, multivalent fucose, glycosylated polyaminoacids and lectins.
  • the term multivalent indicates that more than one monosaccharide unit is present.
  • Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.
  • a number of folate and folate analogs amenable to the present disclosure as ligands are described in U.S. Pat. Nos.2,816,110; 5,552,545; 6,335,434 and 7,128,893, contents of which are herein incorporated in their entireties by reference.
  • the terms “PK modulating ligand” and “PK modulator” refers to molecules which can modulate the pharmacokinetics of the composition of the disclosure.
  • Some exemplary PK modulator include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, and transthyretia- binding ligands (e.g., tetraiidothyroacetic acid, 2, 4, 6-triiodophenol and flufenamic acid).
  • lipophilic molecules bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carpro
  • Oligomeric compounds that comprise a number of phosphorothioate intersugar linkages are also known to bind to serum protein, thus short oligomeric compounds, e.g. oligonucleotides of comprising from about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), and that comprise a plurality of phosphorothioate linkages in the backbone are also amenable to the present disclosure as ligands (e.g. as PK modulating ligands).
  • ligands e.g. as PK modulating ligands
  • the PK modulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and/or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in PK modulating oligonucleotide are phosphorothioate and/or phosphorodithioates linkages.
  • aptamers that bind serum components e.g. serum proteins
  • Docket No.: 121301-22520 ALN-511-WO are also amenable to the present disclosure as PK modulating ligands. Binding to serum components (e.g.
  • ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties.
  • a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties.
  • all the ligands have different properties.
  • a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., monomer- linker-NH 2 can be incorporated into into a component of the compounds of the disclosure (e.g., a dsRNA agent of the disclosure or linker).
  • a ligand having an electrophilic group e.g., a pentafluorophenyl ester or aldehyde group
  • a monomer having a chemical group suitable for taking part in Click Chemistry reaction can be incorporated e.g., an azide or alkyne terminated tether/linker.
  • a ligand having complementary chemical group e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
  • ligand can be conjugated to nucleobases, sugar moieties, or internucleosidic linkages of the dsRNA agent of the disclosure. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety.
  • Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position.
  • the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety.
  • the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing.
  • Conjugation to sugar moieties of nucleosides can occur at any carbon atom.
  • Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms.
  • the 1' position can also be attached to a conjugate moiety, such as in an abasic residue.
  • Internucleosidic linkages can also bear conjugate moieties.
  • the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to Docket No.: 121301-22520 ALN-511-WO the phosphorus atom.
  • the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
  • a reactive group e.g., OH, SH, amine, carboxyl, aldehyde, and the like
  • one reactive group is electrophilic and the other is nucleophilic.
  • an electrophilic group can be a carbonyl-containing functionality and a nucleophilic group can be an amine or thiol.
  • Methods for conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in the literature such as, for example, in Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety.
  • the ligand can be attached to the dsRNA agent of the disclosures via a linker or a carrier monomer, e.g., a ligand carrier.
  • the carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.”
  • a “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier monomer into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of an oligonucleotide.
  • a “tethering attachment point” in refers to an atom of the carrier monomer, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety.
  • the selected moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide.
  • the selected moiety is connected by an intervening tether to the carrier monomer.
  • the carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent atom.
  • a functional group e.g., an amino group
  • another chemical entity e.g., a ligand to the constituent atom.
  • Representative U.S. patents that teach the preparation of conjugates of nucleic acids include, but are not limited to, U.S. Pat.
  • the dsRNA agent further comprises a targeting ligand that targets a liver tissue.
  • the targeting ligand is a carbohydrate-based ligand.
  • the targeting ligand is a GalNAc conjugate.
  • the dsRNA agent of the disclosure further comprises a ligand having a structure shown below: , wherein: L G is independently for each occurrence a ligand, e.g., carbohydrate, e.g.
  • the dsRNA agent of the disclosure comprises a ligand of Formula , Formula (IV) , or Formula (V) , wherein: q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; Q and Q’ are independently for each occurrence is absent, –(P 7 -Q 7 -R 7 )p-T 7 - or –T 7 -Q 7 -T 7’ -B- T 8 and T 8’ are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O
  • the iRNA agent can then contain multiple ligands via the same or different backbone attachment points to the carrier, or via the branched linker(s).
  • the branchpoint of the branched linker may be a bivalent, trivalent, tetravalent, pentavalent ,or hexavalent atom, or a group presenting such multiple valencies.
  • the branchpoint is -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, - N(Q)C(O)-C, or -N(Q)C(O)O-C; wherein Q is independently for each occurrence H or optionally substituted alkyl.
  • the branchpoint is glycerol or glycerol derivative. Docket No.: 121301-22520 ALN-511-WO Suitable ligands for use in the compositions of the disclosure are described in U.S.
  • a suitable ligand is a ligand disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference.
  • the ligand comprises the structure below:
  • the dsRNA of the disclosure a ligand of structure:
  • the dsRNA agent of the disclosure is conjugated with a ligand of structure:
  • the dsRNA agent of the disclosure comprises a ligand of structure: Docket No.: 121301-22520 ALN-511-WO
  • the dsRNA agent of the disclosure comprises a monomer of structure: .
  • the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S Docket No.: 121301-22520 ALN-511-WO .
  • the RNAi agent is conjugated to L96 as defined in Table 1 and shown below: . Synthesis of above described ligands and monomers is described, for example, in US Patent No.8,106,022, content of which are incorporated herein by reference in their entirety. VIII.
  • 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 an ACVR1C-associated disorder, e.g., metabolic disorder
  • delivery 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 (see e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol.2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties).
  • 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.
  • RNAi agent can Docket No.: 121301-22520 ALN-511-WO be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation.
  • Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the RNAi agent to be administered.
  • Several studies have shown successful knockdown of gene products when an RNAi agent is administered locally.
  • pulmonary delivery e.g., inhalation
  • a dsRNA e.g., SOD1
  • Intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ. et al. (2003) Mol. Vis.9:210- 216) were also both shown to prevent neovascularization in an experimental model of age-related macular degeneration.
  • RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther.12:59-66; Makimura, H.
  • RNAi agent for administering a RNAi agent systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the 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.
  • RNAi agent directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an RNAi agent to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol.24:1005-1015).
  • the RNAi agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system.
  • drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system.
  • Positively charged cationic delivery systems facilitate binding of molecule RNAi agent (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell.
  • Cationic lipids, dendrimers, or polymers can either be bound to an RNAi agent, or induced to form a Docket No.: 121301-22520 ALN-511-WO vesicle or micelle (see e.g., Kim SH.
  • RNAi agent encases an RNAi agent.
  • the formation of vesicles or micelles further prevents degradation of the RNAi agent when administered systemically.
  • Methods for making and administering cationic- RNAi agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res.9:1291-1300; Arnold, AS et al. (2007) J. Hypertens.25:197-205, which are incorporated herein by reference in their entirety).
  • RNAi agents include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111- 114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther.12:321-328; Pal, A. et al., (2005) Int J. Oncol.26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A.
  • RNAi agent forms a complex with cyclodextrin for systemic administration.
  • Methods for administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S.
  • Patent No.7, 427, 605 which is herein incorporated by reference in its entirety.
  • Certain aspects of the instant disclosure relate to a method of reducing the expression of an ACVR1C gene in a cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure.
  • the cell is a liver 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, adipose tissue.
  • Another aspect of the disclosure relates to a method of reducing the expression and/or activity of an ACVR1C gene in a subject, comprising administering to the subject the double-stranded RNAi agent of the disclosure.
  • an ACVR1C- associated disorder comprises a metabolic disorder.
  • the metabolic disorder comprises metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight.
  • the double-stranded RNAi agent is administered subcutaneously. In one embodiment, the double-stranded RNAi agent is administered intramuscularly. In one embodiment, the double-stranded RNAi agent is administered by intravenously. In one embodiment, the double-stranded RNAi agent is administered by pulmonary sytem administration, e.g., intranasal administration, or oral inhalative administration. Docket No.: 121301-22520 ALN-511-WO For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to 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, intraventricular, oral, topical, rectal, anal, vaginal, nasal, and ocular.
  • routes include pulmonary system, intravenous, subcutaneous, intraventricular, 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 and a pharmaceutically acceptable carrier.
  • compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be 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, or intrathecal or intraventricular administration.
  • 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.
  • compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration.
  • suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration.
  • Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives.
  • Such administration permits both systemic and local delivery of the double stranded RNAi agents of the disclosure.
  • Intranasal administration may include instilling or insufflating a double stranded RNAi agent into the nasal cavity with syringes or droppers by applying a few drops at a time or via atomization.
  • Suitable dosage forms for intranasal administration include drops, powders, nebulized mists, and sprays.
  • Nasal delivery devices include, but not limited to, vapor inhaler, nasal dropper, spray bottle, metered dose spray pump, gas driven spray atomizer, nebulizer, mechanical powder sprayer, breath Docket No.: 121301-22520 ALN-511-WO actuated inhaler, and insufflator.
  • Devices for delivery deeper into the respiratory system, e.g., into the lung, include nebulizer, pressured metered-dose inhaler, dry powder inhaler, and thermal vaporization aerosol device.
  • Devices for delivery by inhalation are available from commercial suppliers.Devices can be fixed or variable dose, single or multidose, disposable or reusable depending on, for example, the disease or disorder to be prevented or treated, the volume of the agent to be delivered, the frequency of delivery of the agent, and other considerations in the art.
  • Oral inhalative administration may include use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI), to deliver a double stranded RNAi agent to the pulmonary system.
  • DPI passive breath driven or active power driven single/-multiple dose dry powder inhaler
  • Suitable dosage forms for oral inhalative administration include powders and solutions.
  • Suitable devices for oral inhalative administration include nebulizers, metered-dose inhalers, and dry powder inhalers.
  • Dry powder inhalers are of the most popular devices used to deliver drugs, especially proteins to the lungs.
  • Exemplary commercially available dry powder inhalers include Spinhaler (Fisons Pharmaceuticals, Rochester, NY) and Rotahaler (GSK, RTP, NC).
  • nebulizers are available, namely jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers. Jet nebulizers are driven by compressed air.
  • Ultrasonic nebulizers use a piezoelectric transducer in order to create droplets from an open liquid reservoir. Vibrating mesh nebulizers use perforated membranes actuated by an annular piezoelement to vibrate in resonant bending mode.
  • the holes in the membrane have a large cross-section size on the liquid supply side and a narrow cross- section size on the side from where the droplets emerge.
  • the hole sizes and number of holes can be adjusted. Selection of a suitable device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung. Aqueous suspensions and solutions are nebulized effectively. Aerosols based on mechanically generated vibration mesh technologies also have been used successfully to deliver proteins to lungs.
  • RNAi agent for pulmonary system administration may vary from one target gene to another target gene and the appropriate amount that has to be applied may have to be PQ ⁇ Q ⁇ XUYQP UYPU ⁇ UP_MWWc RZ ⁇ QMOT ⁇ M ⁇ SQ ⁇ SQYQ( Gc[UOMWWc& ⁇ TU] MXZ_Y ⁇ ⁇ MYSQ] R ⁇ ZX *) tS ⁇ Z + XS& Z ⁇ .) tS ⁇ Z *.)) tS& Z ⁇ *)) tS ⁇ Z *))) tS(
  • Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • 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.
  • disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
  • compositions suitable for oral Docket No.: 121301-22520 ALN-511-WO administration of the agents of the disclosure are further described in PCT Application No. PCT/US20/33156, the entire contents of which are incorporated herein by reference.
  • Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives.
  • Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives.
  • Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.
  • the total concentration of solutes may be controlled to render the preparation isotonic.
  • the administration of the siRNA compound is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary system, intranasal, urethral, or ocular.
  • Administration can be provided by the subject or by another person, e.g., a health care provider.
  • the medication can be provided in measured doses or in a dispenser which delivers a metered dose.
  • RNAi agents targeting the ACVR1C 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.
  • These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating 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.
  • 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 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 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. Delivery of 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 Docket No.: 121301-22520 ALN-511-WO 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 or avipox, e.g.
  • pox virus vectors such as an orthopox, e.g., vaccinia virus vectors 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 regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the RNAi agent in target cells.
  • regulatory elements e.g., promoters, enhancers, etc.
  • 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 who would benefit from inhibiting or reducing the expression of an ACVR1C gene , e.g., a subject having an ACVR1C-associated disorder, e.g., a subject having or at risk of having or at risk of developing an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight.
  • compositions are formulated based on the mode of delivery.
  • One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery.
  • 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 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 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 Docket No.: 121301-22520 ALN-511-WO 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 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, (
  • 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 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.
  • the oral dosage form of the pharmaceutical composition includes an excipient.
  • the excipient is polyethyleneglycol. In another example the excipient is precirol.
  • the oral dosage form of the pharmaceutical composition includes a plasticizer.
  • the plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.
  • the methods include contacting the cell with a dsRNA of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcripts of an ACVR1C gene, thereby inhibiting expression of the ACVR1C gene in the cell.
  • Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of a target may be determined by determining the mRNA expression level of the target gene using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of a target protein using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques.
  • the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
  • Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible.
  • the cell may be an extra-hepatic cell, such as a liver cell or an adipocyte.
  • a cell suitable for treatment using the methods of the disclosure may be any cell that expresses a target gene.
  • a cell suitable for use in the methods of the disclosure may be a mammalian Docket No.: 121301-22520 ALN-511-WO cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a rat cell, or a mouse cell.
  • the cell is a human cell, e.g., a human liver cell or a human kidney cell.
  • Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art.
  • the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest.
  • the RNAi agent does not include a targeting ligand.
  • inhibiting is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.
  • a level of inhibition e.g., for an RNAi agent of the instant disclosure, can be assessed in cell culture conditions, e.g., wherein cells in cell culture are transfected via Lipofectamine TM -mediated transfection at a concentration in the vicinity of a cell of 10 nM or less, 1 nM or less, etc.
  • Knockdown of a given RNAi agent can be determined via comparison of pre-treated levels in cell culture versus post-treated levels in cell culture, optionally also comparing against cells treated in parallel with a scrambled or other form of control RNAi agent.
  • Knockdown in cell culture of, e.g., 50% or more, can thereby be identified as indicative of “inhibiting” or “reducing”, “downregulating” or “suppressing”, etc. having occurred. It is expressly contemplated that assessment of targeted mRNA or encoded protein levels (and therefore an extent of “inhibiting”, etc. caused by a RNAi agent of the disclosure) can also be assessed in in vivo systems for the RNAi agents of the instant disclosure, under properly controlled conditions as described in the art.
  • an ACVR1C gene or “inhibiting expression of ACVR1C,” as used herein, includes inhibition of expression of any ACVR1C gene (such as, e.g., a mouse ACVR1C gene, a rat ACVR1C gene, a monkey ACVR1C gene, or a human ACVR1C gene) as well as variants or mutants of an ACVR1C gene that encodes an ACVR1C protein.
  • the ACVR1C gene may be a wild-type ACVR1C gene, a mutant ACVR1C gene, or a transgenic ACVR1C gene in the context of a genetically manipulated cell, group of cells, or organism.
  • “Inhibiting expression of an ACVR1C gene” includes any level of inhibition of an ACVR1C gene, e.g., at least partial suppression of the expression of an ACVR1C gene, such as an inhibition by at least 20%. In certain embodiments, inhibition is by at least 30%, at least 40%, at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to below the level of detection of the assay method.
  • the expression of an ACVR1C gene may be assessed based on the level of any variable associated with ACVR1C gene expression, e.g., ACVR1C mRNA level or ACVR1C protein level.
  • Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level.
  • the control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control). Docket No.: 121301-22520 ALN-511-WO
  • expression of an ACVR1C gene is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay.
  • the methods include a clinically relevant inhibition of expression of ACVR1C, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of an ACVR1C gene.
  • Inhibition of the expression of an ACVR1C gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which an ACVR1C gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a RNAi agent of the disclosure, or by administering a RNAi agent of the disclosure to a subject in which the cells are or were present) such that the expression of an ACVR1C gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with a RNAi agent or not treated with a
  • the degree of inhibition may be expressed in terms of: (mRNAincontrolcells) - (mRNAin treated cells) ⁇ 100 % (mRNAincontrol cells)
  • inhibition of the expression of an ACVR1C gene may be assessed in terms of a reduction of a parameter that is functionally linked to an ACVR1C gene expression, e.g., ACVR1C protein expression.
  • ACVR1C gene silencing may be determined in any cell expressing an ACVR1C gene, either endogenous or heterologous from an expression construct, and by any assay known in the art.
  • Inhibition of the expression of an ACVR1C protein may be manifested by a reduction in the level of the ACVR1C protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject).
  • the inhibiton of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.
  • a control cell or group of cells that may be used to assess the inhibition of the expression of an ACVR1C gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the disclosure.
  • control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.
  • the level of ACVR1C mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing RNA expression.
  • the level of expression of ACVR1C in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the ACVR1C gene.
  • RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy TM RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland).
  • Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on Docket No.: 121301-22520 ALN-511-WO assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating ACVR1C mRNA may be detected using methods the described in WO2012/177906, the entire contents of which are hereby incorporated herein by reference.
  • the level of expression of ACVR1C is determined using a nucleic acid probe.
  • probe refers to any molecule that is capable of selectively binding to a specific ACVR1C nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays.
  • PCR polymerase chain reaction
  • RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to ACVR1C RNA.
  • the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an Affymetrix ® gene chip array.
  • a skilled artisan can readily adapt known RNA detection methods for use in determining the level of ACVR1C mRNA.
  • An alternative method for determining the level of expression of ACVR1C in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, US Patent No.4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci.
  • the level of expression of ACVR1C is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan TM System), by a Dual-Glo® Luciferase assay, or by other art-recognized method for measurement of ACVR1C expression or mRNA level.
  • the expression level of ACVR1C mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See US Patent Nos.
  • the determination of ACVR1C expression level may also comprise using nucleic acid probes in solution. Docket No.: 121301-22520 ALN-511-WO
  • the level of RNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of ACVR1C nucleic acids.
  • the level of ACVR1C protein expression may be determined using any method known in the art for the measurement of protein levels.
  • Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme- linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.
  • assays can also be used for the detection of proteins indicative of the presence or replication of ACVR1C proteins.
  • the efficacy of the methods of the disclosure in the treatment of an ACVR1C-related disease is assessed by a decrease in ACVR1C mRNA level (e.g, by assessment of a blood ACVR1C gene level, or otherwise). In some embodiments, the efficacy of the methods of the disclosure in the treatment of an ACVR1C-related disease is assessed by a decrease in ACVR1C mRNA level (e.g, by assessment of a liver or kidney sample for ACVR1C level, by biopsy, or otherwise). In some embodiments of the methods of the disclosure, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject.
  • the inhibition of expression of ACVR1C may be assessed using measurements of the level or change in the level of ACVR1C mRNA or ACVR1C protein in a sample derived from a specific site within the subject, e.g., liver or kidney cells.
  • the methods include a clinically relevant inhibition of expression of ACVR1C, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of ACVR1C.
  • the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present.
  • methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.
  • XI. Prophylactic and Treatment Methods of the Disclosure The present disclosure also provides methods of using an iRNA of the disclosure or a composition containing an iRNA of the disclosure to inhibit expression of ACVR1C, thereby preventing or treating an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight.
  • an ACVR1C-associated disorder e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g
  • the cell may be contacted with the siRNA in vitro or in vivo, i.e., the cell may be within a subject.
  • a cell suitable for treatment using the methods of the disclosure may be any cell that expresses an ACVR1C gene, e.g., an adipocyte cell, or a liver cell.
  • a cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell, including human cell in a chimeric non-human animal, or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), or a non-primate cell.
  • the cell is a human cell, e.g., a human liver cell.
  • ACVR1C gene expression is inhibited in the cell by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or to a level below the level of detection of the assay.
  • the in vivo methods of the disclosure may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ACVR1C gene of the mammal to which the RNAi agent is to be administered.
  • composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, intraocular (e.g., periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, anterior or posterior juxtascleral, subretinal, subconjunctival, retrobulbar, or intracanalicular injection), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), and topical (including buccal and sublingual) administration.
  • intracranial e.g., intraventricular, intraparenchymal, and intrathecal
  • intravenous intramuscular, subcutaneous, transdermal, airway (aerosol)
  • nasal rectal
  • intraocular e.g.,
  • the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by intramuscular injection.
  • the mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.
  • the present disclosure also provides methods for inhibiting the expression of an ACVR1C gene in a mammal.
  • the methods include administering to the mammal a composition comprising a dsRNA that targets an ACVR1C gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the ACVR1C gene, thereby inhibiting expression of the ACVR1C gene in the cell.
  • Reduction in gene expression can be assessed by any methods known in the art and by methods, e.g. qRT-PCR, described herein, e.g., in Example 2.
  • Reduction in protein production can be assessed by any methods known it the art, e.g. ELISA.
  • a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the target gene or protein expression.
  • a blood sample serves as the subject sample for monitoring the reduction in the target protein expression.
  • the present disclosure further provides methods of treatment in a subject in need thereof, e.g., a subject diagnosed with an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight. Docket No.: 121301-22520 ALN-511-WO The present disclosure further provides methods of prophylaxis in a subject in need thereof.
  • the treatment methods of the disclosure include administering an iRNA of the disclosure to a subject, e.g., a subject that would benefit from a reduction of expression of an ACVR1C gene, in a prophylactically effective amount of a dsRNA targeting ACVR1C or a pharmaceutical composition comprising a dsRNA targeting ACVR1C.
  • the present disclosure provides methods of treating a subject having a disorder that would benefit from reduction in expression of an ACVR1C gene, e.g., an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., diabetes.
  • Treatment of a subject that would benefit from a reduction and/or inhibition of ACVR1C gene expression includes therapeutic treatment (e.g., a subject is having an ACVR1C-associated disorder, e.g., a metabolic disorder) and prophylactic treatment (e.g., the subject is not having an ACVR1C-associated disorder, or a subject may be at risk of developing an ACVR1C-associated disorder).
  • the an ACVR1C-associated disorder is a metabolic disorder.
  • metablic disorder examples include but are not limited to, metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight.
  • the metablic disorder is metabolic syndrome.
  • the RNAi agent is administered to a subject in an amount effective to inhibit expression of ab activin A receptor type 1C (ACVR1C) gene in a cell within the subject.
  • ACVR1C ab activin A receptor type 1C
  • the amount effective to inhibit ACVR1C expression in a cell within a subject may be assessed using methods discussed above, including methods that involve assessment of the inhibition of ACVR1C mRNA, ACVR1C protein, or related variables, such as insulin resistance, BMI, WHRadj BMI, adipose tissue, e.g., image-based quantification of adipose tissue, e.g., MRI or DEXA for abdominal subcutaneous adipose and visceral adipose tissue quantification.
  • An iRNA of the disclosure may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition.
  • the naked iRNA may be in a suitable buffer solution.
  • the buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
  • the buffer solution is phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.
  • an iRNA of the disclosure may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.
  • Subjects that would benefit from an inhibition of ACVR1C gene expression are subjects susceptible to or diagnosed with an ACVR1C-associated disorder, e.g., a metablic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight.
  • the method includes administering a composition featured herein such that expression of the target gene is decreased, such Docket No.: 121301-22520 ALN-511-WO as for about 1, 2, 3, 4, 5, 6, 1-6, 1-3, or 3-6 months per dose.
  • the composition is administered once every 3-6 months.
  • the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the ACVR1C gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.
  • Administration of the iRNA according to the methods of the disclosure may result prevention or treatment of an ACVR1C-associated disorder, e.g., a metablic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight.
  • Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg.
  • the iRNA is administered subcutaneously, i.e., by subcutaneous injection.
  • One or more injections may be used to deliver the desired dose of iRNA to a subject.
  • the injections may be repeated over a period of time.
  • the administration may be repeated on a regular basis.
  • the treatments can be administered on a less frequent basis.
  • a repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as once per month to once a year.
  • the iRNA is administered about once per month to about once every three months, or about once every three months to about once every six months.
  • the disclosure further provides methods and uses of an iRNA agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of ACVR1C gene expression, e.g., a subject having an ACVR1C-associated disorder, e.g., a metabolic disorder, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders.
  • the methods which include administration of an iRNA agent of the disclosure further include administering to the subject one or more additional therapeutic agents.
  • a dsRNA targeting ACVR1C is administered in combination with, e.g., an agent useful in treating an ACVR1C-associated disorder, e.g., a metabolic disorder, as described herein or otherwise known in the art.
  • an agent useful in treating an ACVR1C-associated disorder e.g., a metabolic disorder
  • additional agents and treatments suitable for treating a subject that would benefit from reducton in ACVR1C expression e.g., a subject having an ACVR1C-associated disorder, e.g., a metabolic disorder, may include agents currently used to treat symptoms of an ACVR1C-associated disorder.
  • dsRNA agent of the disclosure examples include, but are not limited to, insulin, a glucagon-like peptide 1 agonist (e.g., exenatide, liraglutide, dulaglutide, semaglutide, and pramlintide), a sulfonylurea (e.g., chlorpropamide, glipizide), a seglitinide (e.g., repaglinide, nateglinidie), biguanides (e.g., metformin), a Docket No.: 121301-22520 ALN-511-WO thiazolidinedione, e.g, rosiglitazone, troglitazone, an alpha-glucosidase inhibitor (e.g., acarbose and meglitol ), an SGLT2 inhibitor (e.g., dapagliflozin), a DPP-4 inhibitor (e.
  • Additional therapeutic agents can also include other siRNAs targeting the liver (e.g. INHBE).
  • the iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.
  • XII siRNAs targeting the liver
  • kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a siRNA compound, or a DNA which encodes an siRNA compound, e.g., a double- stranded siRNA compound, or ssiRNA compound, or precursor thereof).
  • a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a siRNA compound, or a DNA which encodes an siRNA compound, e.g., a double- stranded siRNA compound, or ssiRNA compound, or precursor thereof).
  • Such kits include one or more dsRNA agent(s) and instructions for use,
  • the dsRNA agent may be in a vial or a pre-filled syringe.
  • the kits may optionally further comprise means for administering the dsRNA agent (e.g., an injection device, such as a pre-filled syringe), or means for measuring the inhibition of an ACVR1C gene (e.g., means for measuring the inhibition of ACVR1C mRNA, ACVR1C protein, and/or ACVR1C activity).
  • Such means for measuring the inhibition of ACVR1C gene may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample.
  • the kits of the disclosure may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount.
  • the individual components of the pharmaceutical formulation may be provided in one container, e.g., a vial or a pre-filled syringe.
  • the kit may be packaged in a number of different configurations such as one or more containers in a single box.
  • the different components can be combined, e.g., according to instructions provided with the kit.
  • the components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition.
  • the kit can also include a delivery device.
  • siRNA Design siRNAs targeting the activin A receptor type 1C (ACVR1C) gene (ACVR1C, human: NCBI refseqID NM_145259.3, NCBI Gene ID: 130399) were designed using custom R and Python scripts.
  • the human NM_145259.3 mRNA has a length of 8853 bases.
  • Table 2 Detailed lists of the unmodified sense and antisense strand sequences of ACVR1C dsRNA agents are shown in Table 2.
  • Detailed lists of the modified sense and antisense strand sequences of ACVR1C dsRNA agents are shown in Table 3.
  • siRNA Synthesis siRNAs were designed, synthesized, and prepared using methods known in the art. Briefly, as an exemplary method, siRNA sequences were synthesized on a 1 ⁇ mol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on solid supports. Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramidite monomers (2’- deoxy-2’-fluoro, 2’-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, WI), Hongene (China), or Chemgenes (Wilmington, MA, USA).
  • phosphoramidite monomers were procured from commercial suppliers, prepared in-house, or procured using custom synthesis from various CMOs.
  • Phosphoramidites were prepared at a concentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMF and were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) with a reaction time of 400 s.
  • ETT 5-Ethylthio-1H-tetrazole
  • the lipophilic moiety e.g., the exemplary C 22 conjugate
  • solid-supported oligoribonucleotides were treated with 300 ⁇ L of Methylamine (40% aqueous) at room temperature in 96 well plates for approximately 2 hours to afford cleavage from the solid support and subsequent removal of all additional base-labile protecting groups.
  • Methylamine 50% aqueous
  • TDMS tert-butyl dimethyl silyl
  • oligonucleotide solution in aqueous methylamine was added 200 ⁇ L of dimethyl sulfoxide (DMSO) and 300 ⁇ L TEA.3HF and the solution was incubated for approximately 30 mins at 60 °C. After incubation, the plate was allowed to come to room temperature and crude oligonucleotides were precipitated by the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1 ethanol:isopropanol. The plates were then centrifuged at 4 °C for 45 mins and the supernatant carefully decanted with the aid of a multichannel pipette.
  • DMSO dimethyl sulfoxide
  • the oligonucleotide pellet was resuspended in 20 mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5 mL, GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector. Desalted samples were collected in 96 well plates and then analyzed by LC-MS and UV spectrometry to confirm identity and quantify the amount of material, respectively. Duplexing of single strands was performed on a Tecan liquid handling robot.
  • Sense and antisense single strands were combined in an equimolar ratio to a final concentration of 10 ⁇ M in 1x PBS in 96 well plates, the plate sealed, incubated at 100 °C for 10 minutes, and subsequently allowed to return slowly to room temperature over a period of 2-3 hours. The concentration and identity of each duplex was confirmed and then subsequently utilized for in vitro screening assays.
  • Example 2
  • SK-MEL-28 cells (Cat # HTB-72, ATCC, Manassas, VA) were grown to near confluence at 37°C in an atmosphere of 5% CO 2 in Eagle’s Minimum Essential Medium (Gibco) supplemented with 10% FBS (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 5 ql of Opti-MEM plus 0.1 ql of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA. cat # 13778-150) to 5 ql of each siRNA duplex to an individual well in a 384-well plate. The mixture was then incubated at room temperature for 15 minutes.
  • RNA isolation using DYNABEADS mRNA Isolation Kit (InvitrogenTM, part #: 610-12) Docket No.: 121301-22520 ALN-511-WO RNA was isolated using an automated protocol on a BioTek-EL406 platform using DynabeadsTM mRNA DIRECTTM Purification Kit (InvitrogenTM, Catalog No.61012).
  • cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, Cat #4374967)
  • cDNA Complementary DNA
  • a master mix containing 1 ⁇ L 10X Buffer, 0.4 ⁇ L 25X deoxyribonucleotide triphosphate, 1 ⁇ L 10X Random primers, 0.5 ⁇ L Reverse Transcriptase, 0.5 ⁇ L RNase inhibitor, and 6.6 ⁇ L of water per reaction was added to RNA isolated above.
  • the sense and antisense sequences of AD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14964) and antisense UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 14965).
  • Table 6 shows the results of three dose screens in SK-MEL-28 cells transfected with the indicated agents from Tables 2 and 3.
  • Table 1. Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds; and it is understood that when the nucleotide contains a 2’- fluoro modification, then the fluoro replaces the hydroxy at that position in the parent nucleotide (i.e., it is a 2’-deoxy-2’-fluoronucleotide).
  • nucleotide abbreviations in the table omit the 3’-phosphate (i.e., they are 3’-OH) when placed at the 3’-terminal position of an oligonucleotide.
  • siRNAs targeting the mouse activin A receptor type 1C (ACVR1C) gene (NCBI refseqID NM_001033369.3, NCBI Gene ID: 269275) or the human ACVR1C gene (NCBI refseqID NM_145259.3, NCBI Gene ID: 130399) were designed using custom R and Python scripts.
  • the human NM_145259.3 mRNA has a length of 8853 bases.
  • the mouse NM_001033369.3 mRNA has a length of 8666 bp.

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Abstract

The present disclosure relates to RNAi agents, e.g., double stranded RNA (dsRNA) agents, targeting an activin A receptor type 1C (ACVR1C) gene. The disclosure also relates to methods of using such RNAi agents to inhibit expression of an ACVR1C gene and to methods of preventing and treating an ACVRIC-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome.

Description

Docket No.: 121301-22520 ALN-511-WO ACTIVIN A RECEPTOR TYPE 1C (ACVR1C) iRNA COMPOSITIONS AND METHODS OF USE THEREOF RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No.63/539,682, filed on September 21, 2024, and U.S. Provisional Application No.63/694,377, filed on September 13, 2024. The entire contents of each of the foregoing applications are incorporated herein by reference. SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on September 16, 2024, is named “121301_22520_SL.xml” and is 84,670,000 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE DISCLOSURE With the successful development of treatmeants for many infectious diseases in most of the world, non-communicable diseases, metabolic disorders in particular, have become a major health hazard of the modern world. The increase in consumption of high calorie-low fiber fast food and the decrease in physical activity due to mechanized transportations and sedentary lifestyle have resulted in the spread of obesity and metabolic disorders such as metabolic syndrome, type 2 diabetes, hypertension, cardiovascular diseases, stroke, and other disabilities. Indeed, the occurences of subjects with a metabolic disorder, such as, metabolic syndrome that encompasses a number of health conditions placing them at higher risk for heart disease, diabetes, stroke, and other diseases have increased in the recent years. Activin A receptor type 1C (ACVR1C, which encodes an activitn-receptor like kinase 7 [\Z^QUY& 5@?0$ U] M ^c[Q = \QOQ[^Z\ RZ\ ^TQ G;:'q RMXUWc ZR ]USYMWUYS XZWQO_WQ( 57HE*7 TM] intrinsic serine/threonine kinase activities in its cytoplasmic domains, inducing phosphorylation and activation of the SMAD2/3/4 complex, which translocates into the nucleus where it binds SMAD- binding elements to regulate gene transcription. Expression levels of ACVR1C vary greatly among tissues, but white and brown adipose tissues have the highest level of expression. ACVR1C functions to antagonize catabolic pathways of lipolysis and amino acid degradation to facilitate the preservation and accumulation of energy stores. A polymorphism in ACVR1C has been found to be associated with increased risk of metabolic syndrome in Chinese females and may be involved in cardiovascular remodeling in patients with metabolic syndrome (Zhang, W et al. Arq Bras Cardiol.2013: 101(2):134-140). Additionally, variants predicted to lead to loss of ACVR1C gene function are thought to influence body fat distribution and protect against type 2 diabetes (Emdin CA et al. Diabetes.2019: 68(1):226-234; Justice AE et al. Nat Genet.2019: 51(3):452-69; Koprulu M Docket No.: 121301-22520 ALN-511-WO et al. The Journal of Clincal Endocrinology & Metabolism.2022: 107(4):1065-1077; Deaton AM et al. Nature Communications.2022: 13:4319; Akbari P et al. Nature Communications.202213:4844). A study conducted in adipocytes of an obese mouse strain demonstrated that ACVR1C dysfunction (due to a nonsense mutation) caused increased lipolysis in adipocytes and led to decreased fat accumulation, and conversely, ACVR1C activation inhibited lipolysis by suppressing the expression of adipose lipases (Guo T et al. eLife 2014.1-18 (DOI: 10.7554/eLife.03245; Adam RC et al. Proc. Natl. Acad. Sci. U.S.A.2023.120(32)). Furthermore, ACVR1C-deficient mice with lower body weight exhibited enhanced glucose tolerance and insulin sensitivity, and measurement of metabolic rates in these mice revealed increased O2 consumption, decreased respiratory quotients, and increased energy expenditure (Yogosawa, S et al. Diabetes 2013: 62(1):115-123). These findings suggest that specific inhibitors of ACVR1C may have therapeutic applications in metabolic disorders, such as obesity and metabolic syndrome. Current treatments for disorders of metabolic disorders include lifestyle changes, dieting, exercise and treatment with agents, such as lipid lowering agents, e.g., statins, incretin agonists, and other drugs. However, these therapies and treatments are often limited by compliance, are not always effective (sometimes due to patient adherence), result in side effects, and result in drug-drug interactions. Accordingly, there is a need in the art for alternative treatments for subjects having metabolic disorders, such as obesity and metablic syndrome and related diseases, e.g., diabetes, hypertension, and cardiovascular disease, such as an agent that can selectively and efficiently silence an activin A receptor type 1C (ACVR1C) gene, using the cell’s own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of the ACVR1C gene. SUMMARY OF THE DISCLOSURE The present disclosure provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a target gene encoding activin A receptor type 1C (ACVR1C). The ACVR1C 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 an ACVR1C gene, and/or for treating a subject who would benefit from inhibiting or reducing the expression of an ACVR1C gene, e.g., a subject suffering or prone to suffering from an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, diabetes, hypertension, and/or cardiovascular disease. Accordingly, in an aspect, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from the nucleotide sequence of SEQ ID NO:2 and wherein one or more C22 hydrocarbon chains are conjugated to the sense strand. Docket No.: 121301-22520 ALN-511-WO In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 In another aspect, the disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte and/or a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence of any one of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from the corresponding portion of the nucleotide sequence of any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28. In another aspect, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte and/or a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding ACVR1C, and wherein the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. In yet another aspect, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte and/or a liver cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the sense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. In some embodiments, these dsRNA agents further comprise one or more C22 hydrocarbon chains conjugated to one or more positions, e.g., internal positions, on at least one strand of the dsRNA agent. In another aspect, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, such as an adipocyte, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the sense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no Docket No.: 121301-22520 ALN-511-WO more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. In one embodiment, the dsRNA agent comprises a sense strand comprising a contiguous nucleotide sequence which has at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, nucleotide sequence identity over its entire length to any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising a contiguous nucleotide sequence which has at least 85%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, nucleotide sequence identity over its entire length to any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12. In one embodiment, the dsRNA agent comprises a sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than three nucleotides from any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides differing by no more than three nucleotides from any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12. In one embodiment, the dsRNA agent comprises a sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than two nucleotides from any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, or 23 contiguous nucleotides differing by no more than two nucleotides from any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12. In one embodiment, the dsRNA agent comprises a sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than one nucleotide from any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides differing by no more than one nucleotide from any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12. In one embodiment, the dsRNA agent comprises a sense strand comprising or consisting of a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences of the sense strands in any one of Tables 2-5, 7, 8, 11, and 12 and an antisense strand comprising or consisting of a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences of the antisense strands in any one of Tables 2-5, 7, 8, 11, and 12. In an aspect, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding ACVR1C, and wherein the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, Docket No.: 121301-22520 ALN-511-WO contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. In one embodiment, the sense strand comprises at least 15, e.g., 15, 16, 17, 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 2-5, 7, 8, 11, and 12, and the antisense strand comprises at least 15, e.g.,15, 16, 17, 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 2-5, 7, 8, 11, and 12. In some embodiments, the antisense strand comprises at least 15, e.g.,15, 16, 17, 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 of a duplex selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, and AD-2508176. In some embodiments, the sense strand and the antisense strand comprises at least 15, e.g.,15, 16, 17, 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 of a duplex selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, or AD-2508176. In another aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the antisense strand nucleotide sequences selected from the group consisting of (a) 5’-UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’-UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’-UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’-UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. In some embodiments, the sense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides and the antisense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 3, e.g., 3, 2, 1, or 0, nucleotides from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) 5’- AAGCCUAAUGAUGAUAAUUAA-3’ of SEQ ID NO: 14909 and 5’- UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-AUAUCUCAACUUUGUGUCAAA-3’ of SEQ ID NO: 14910 and 5’- UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’- CUAAUGAUGAUAAUUAUGUUA-3’ of SEQ ID NO: 14913 and 5’- UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; Docket No.: 121301-22520 ALN-511-WO (d) 5’- UGUGAAUAUCUUUGAGUCCUA-3’ of SEQ ID NO: 7805 and 5’- UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’- GUGUGUAUGUCUUUUGUGUGA-3’ of SEQ ID NO: 7470 and 5’- UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. In one embodiment, the dsRNA agent comprises at least one modified nucleotide. In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In one embodiment, all of the nucleotides of the sense strand are modified nucleotides; all of the nucleotides of the antisense strand are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3’-terminal deoxythimidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2’-amino-modified nucleotide, a 2’-O-allyl-modified nucleotide, 2’-C-alkyl-modified nucleotide, 2’-hydroxly-modified nucleotide, a 2’-methoxyethyl modified nucleotide, a 2’-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non- natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5’-phosphate, a nucleotide comprising a 5’-phosphate mimic, a thermally destabilizing nucleotide, a glycol modified nucleotide (GNA), a nucleotide comprising a 2’ phosphate, and a 2-O- (N-methylacetamide) modified nucleotide; and combinations thereof. In one embodiment, at least one of the modified nucleotides is selected from the group 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. In one embodiment, 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, a 2'-deoxy-modified nucleotide, a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, a nucleotide with a 2’ phosphate, e.g., G2p, C2p, A2p or U2p, a nucleotide comprising a phosphorothioate group, and a vinyl-phosphonate nucleotide; and combinations thereof. In another embodiment, at least one of the modified nucleotides is a nucleotide with a thermally destabilizing nucleotide modification. In one embodiment, the thermally destabilizing nucleotide modification is 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). Docket No.: 121301-22520 ALN-511-WO In some embodiments, the modified nucleotide comprises a short sequence of 3’-terminal deoxythimidine nucleotides (dT). In some embodiments, the dsRNA agents further comprise a phosphate or phosphate mimic at the 5’-end of the antisense strand. In some embodiments, phosphate mimic is a 5’-vinyl phosphonate (VP). In some embodiments, the 5’-end of the antisense strand of the dsRNA agent does not contain a 5’-vinyl phosphonate (VP). In some embodiments, the dsRNA agent further comprises at least one terminal, chiral phosphorus atom. A site specific, chiral modification to the internucleotide linkage may occur at the 5’ end, 3’ end, or both the 5’ end and 3’ end of a strand. This is being referred to herein as a “terminal” chiral modification. The terminal modification may occur at a 3’ or 5’ terminal position in a terminal region, e.g., at a position on a terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a strand. A chiral modification may occur on the sense strand, antisense strand, or both the sense strand and antisense strand. Each of the chiral pure phosphorus atoms may be in either Rp configuration or Sp configuration, and combination thereof. More details regarding chiral modifications and chirally-modified dsRNA agents can be found in PCT/US18/67103, entitled “Chirally-Modified Double-Stranded RNA Agents,” filed December 21, 2018, which is incorporated herein by reference in its entirety. In some embodiments, the dsRNA agent further comprises a terminal, chiral modification occuring at the first internucleotide linkage at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration. In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occuring at the first, second, and third internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having Docket No.: 121301-22520 ALN-511-WO the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the third internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first, and second internucleotide linkages at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration. In some embodiments, 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, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl. In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3’-terminus of one strand, e.g., the antisense strand or the sense strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5’-terminus of one strand, e.g., the antisense strand or the sense strand. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5’- and 3’-terminus of one strand. In one embodiment, the strand is the antisense strand. In one embodiment, the base pair at the 1 position of the 5^-end of the antisense strand of the duplex is an AU base pair. 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. In one embodiment, each strand is independently no more than 30 nucleotides in length. In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. The region of complementarity may be at least 17 nucleotides in length; between 19 and 23 nucleotides in length; or 19 nucleotides in length. In one embodiment, at least one strand comprises a 3’ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3’ overhang of at least 2 nucleotides. In some embodiments, one or more C22 hydrocarbon chains is conjugated to one or more internal positions on at least one strand of the dsRNA agent. Docket No.: 121301-22520 ALN-511-WO In some embodiments, the lipophilicity of the one or more C22 hydrocarbon chain, measured by octanol-water partition coefficient, logKow, exceeds 0. The lipophilic moiety may possess a logKow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. In some embodiments, the hydrophobicity of the dsRNA agent, measured by the unbound fraction in the plasma protein binding assay of the dsRNA agent, exceeds 0.2. In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the dsRNA agent, measured by fraction of unbound dsRNA 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. The C22 hydrocarbon chain may be saturated or unsaturated. The C22 hydrocarbon chain may be linear or branched In some embodiments, the internal positions include all positions except the three terminal positions from each end of the at least one strand. In some embodiments, the internal positions exclude a cleavage site region of the sense strand. In some embodiments, the internal positions exclude positions 9-12 or positions 11-13, counting from the 5’-end of the sense strand. In some embodiments, the internal positions exclude a cleavage site region of the antisense strand. In some embodiments, the internal positions exclude positions 12-14, counting from the 5’-end of the antisense strand. In some embodiments, the one or more C22 hydrocarbon chains are conjugated to 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 some embodiments, the one or more C22 hydrocarbon chains are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’-end of each strand. In some embodiments, the one or more C22 hydrocarbon chains are conjugated to position 6 on the sense strand, counting from the 5’-end of the sense strand. In some embodiments, the one or more C22 hydrocarbon chains are conjugated to position 16 on the sense strand, counting from the 5’-end of the sense strand. In some embodiments, the one or more C22 hydrocarbon chains is an aliphatic, alicyclic, or polyalicyclic compound, e.g., the one or more C22 hydrocarbon chains contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. In some embodiments, the one or more C22 hydrocarbon chains is a C22 acid, e.g. the C22 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. Docket No.: 121301-22520 ALN-511-WO
Figure imgf000011_0001
In some embodiments, the one or more C22 hydrocarbon chains is a C22 alcohol, e.g., the C22 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, and cis-4,7,10,13,16,19-docosahexanol.
Figure imgf000011_0002
In some embodiments, the one or more C22 hydrocarbon chains is a C22 amide, e.g., the C22 amide is selected 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 one or more C22 hydrocarbon chains may be conjugated to the dsRNA agent via a direct attachment to the ribosugar of the dsRNA agent. Alternatively, the the one or more C22 hydrocarbon chains may be conjugated to the dsRNA agent via a linker or a carrier. In some embodiments, the one or more C22 hydrocarbon chains may be conjugated to the dsRNA agent via internucleotide phosphate linkage.
Figure imgf000011_0003
Docket No.: 121301-22520 ALN-511-WO In certain embodiments, the one or more C22 hydrocarbon chains is conjugated to the dsRNA agent via one or more linkers (tethers), e.g., a carrier that replaces one or more nucleotide(s) in the internal position(s). In some embodiments, the one or more C22 hydrocarbon chains is conjugated to the dsRNA agent via a linker 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. In some embodiments, at least one of the linkers (tethers) is a redox cleavable linker (such as a reductively cleavable linker; e.g., a disulfide group), an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavable linker (e.g., an ester group), a phosphatase cleavable linker (e.g., a phosphate group), or a peptidase cleavable linker (e.g., a peptide bond). In other embodiments, at least one of the linkers (tethers) is a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof. In certain embodiments, the one or more C22 hydrocarbon chains is conjugated to the dsRNA agent via a carrier that replaces one or more nucleotide(s). The carrier can be a cyclic group or an acyclic group. In one embodiment, 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. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone. In some embodiments, the carrier replaces one or more nucleotide(s) in the internal position(s) of the dsRNA agent. In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a receptor which mediates delivery to adipose tissue. In one embodiment, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor related protein (LRP) ligand, bEnd.3 cell binding ligand, transferrin receptor (TfR) ligand, manose receptor ligand, glucose transporter protein, LDL receptor ligand, trans-retinol, RGD peptide, LDL receptor ligand, CD63 ligand, and carbohydrate based ligand. In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a liver tissue. In one embodiment, the targeting ligand is conjugated to the 3’ end of the sense strand of the dsRNA agent. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the targeting ligand is an N-acetylgalactosamine (GalNAc) derivative. In one embodiment, the targeting ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker. In one embodiment, the targeting ligand is Docket No.: 121301-22520 ALN-511-WO
Figure imgf000013_0001
one embodiment, the dsRNA agent is conjugated to the targeting ligand as shown in the following schematic
Figure imgf000013_0002
embodiment, the X is O. In some embodiments, the one or more C22 hydrocarbon chains or targeting ligand is conjugated via a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, funtionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof. In an aspect, the present disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides differing by no more than 4, e.g., 4, 3, 2, 1, or 0, bases and the antisense strand comprises at least 15, e.g.,15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 4, e.g., 4, 3, 2, 1, or 0, bases from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) 5’-asasgcc(Uda)AfaUfGfAfugauaauusasa-3’ of SEQ ID NO: 14933 and 5’- VPusUfsaadTu(Agn)ucaucaUfuAfggcuususg-3’ of SEQ ID NO: 14945; (b) 5’-asusauc(Uda)CfaAfCfUfuugugucasasa-3’ of SEQ ID NO: 14934 and 5’- VPusUfsugdAc(Agn)caaaguUfgAfgauausasg-3’ of SEQ ID NO: 14946; (c) 5’-csusaaugAfuGfAfUfaauu(Ada)ugususa-3’ of SEQ ID NO: 14937 and 5’- VPusAfsacaUfaauuaucAfuCfauuagsgsc-3’ of SEQ ID NO: 14949; Docket No.: 121301-22520 ALN-511-WO (d) 5’-usgsuga(Ada)UfaUfCfUfuugaguccsusa-3’ of SEQ ID NO: 10777 and 5’- VPusAfsggaCfucaaagaUfaUfucacasusu-3’ of SEQ ID NO: 12263; and (e) 5’-gsusgug(Uda)AfuGfUfCfuuuugugusgsa-3’ of SEQ ID NO: 10442 and 5’- VPusCfsacaCfaaaagacAfuAfcacacsusu-3’ of SEQ ID NO: 11928. In one embodiment, a dsRNA agent of the invention is a pharmaceutically acceptable salt thereof. “Pharmaceutically acceptable salts” of each of RNAi agents herein include, but are not limited to, a sodium salt, a calcium salt, a lithium salt, a potassium salt, an ammonium salt, a magnesium salt, and mixtures thereof. One skilled in the art will appreciate that the RNAi agent, when provided as a polycationic salt having one cation per free acid group of the optionally modified phosophodiester backbone and/or any other acidic modifications (e.g., 5’-terminal phosphonate groups). For example, an oligonucleotide of “n” nucleotides in length contains n-1 optionally modified phosophodiesters, so that an oligonucleotide of 21 nt in length may be provided as a salt having up to 20 cations (e.g, 20 sodium cations). Similarly, an RNAi agentshaving a sense strand of 21 nt in length and an antisense strand of 23 nt in length may be provided as a salt having up to 42 cations (e.g, 42 sodium cations). In the preceding example, where the RNAi agent also includes a 5’-terminal phosphate or a 5’-terminal vinylphosphonate group, the RNAi agent may be provided as a salt having up to 44 cations (e.g, 44 sodium cations). The present disclosure also provides cells containing any of the dsRNA agents of the disclosure, and pharmaceutical compositions comprising any of the dsRNA agents of the disclosure. The pharmaceutical composition of the disclosure may include dsRNA agent in an unbuffered solution, e.g., saline or water, or the pharmaceutical composition of the disclosure may include the dsRNA agent is in a buffer solution, e.g., a buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof; or phosphate buffered saline (PBS). In one aspect, the present disclosure provides a method of inhibiting expression of an activin A receptor type 1C (ACVR1C) gene in a cell, such as an adipocyte and/or a liver cell. The method includes contacting the cell with any of the dsRNAs of the disclosure or any of the pharmaceutical compositions of the disclosure, thereby inhibiting expression of the ACVR1C gene in the cell. In one embodiment, the cell is within a subject, e.g., a human subject, e.g., a subject having a metabolic disorder, such as diabetes, metabolic syndrome, cardiovascular disease, or hypertension. In one embodiment, the cell is an adipocyte. In one embodiment, the cell is a hepatocyte. In certain embodiments, the expression of the ACVR1C gene is inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one embodiment, inhibiting expression of the ACVR1C gene decreases ALK7 protein level in serum of the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one aspect, the present disclosure provides a method of treating a subject having a disorder that would benefit from reduction in ALK7 expression. The method includes administering to the subject a therapeutically effective amount of any of the dsRNAs of the disclosure or any of the pharmaceutical compositions of the disclosure, thereby treating the subject having the disorder that would benefit from reduction in ACVR1C expression. Docket No.: 121301-22520 ALN-511-WO In another aspect, the present disclosure provides a method of preventing at least one symptom in a subject having a disorder that would benefit from reduction in ACVR1C expression. The method includes administering to the subject a prophylactically effective amount of any of the dsRNAs of the disclosure or any of the pharmaceutical compositions of the disclosure, thereby preventing at least one symptom in the subject having the disorder that would benefit from reduction in ACVR1C expression. In certain embodiments, the disorder is an ACVR1C-associated disorder. In some embodiments, the ACVR1C-associated disorder is a metabolic disorder. The metabolic disorder may be, e.g. metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight. In some embodiments, the ACVR1C-associated disorder is metabolic syndrome. In some embodiments, the ACVR1C-associated disorder is cardiovascular disease. In some embodiments, the ACVR1C-associated disorder is hypertension. In some embodiments, the ACVR1C-associated disorder is kidney disease. In one embodiment, the subject is human. In one embodiment, administration of the dsRNA to the subject descreases the waist-to-hip ratio adjusted for body mass index in the subject. In certain embodiments, administration of the dsRNA to the subject causes a decrease ALK7 protein accumulation in the subject. In one embodiment, the dsRNA agent is administered to the subject subcutaneously. In one embodiment, the methods of the disclosure include further determining the level of ACVR1C in a sample(s) from the subject. In one embodiment, the level of ACVR1C in the subject sample(s) is an ALK7 protein level in a blood or serum or adipose tissue sample(s). In certain embodiments, the methods of the disclosure further comprise administering to the subject an additional therapeutic agent. In certain embodiments, the additional therapeutic agent is selected from the group consisting of insulin, a glucagon-like peptide 1 agonist, a sulfonylurea, a seglitinide, a biguanide, a thiazolidinedione, an alpha-glucosidase inhibitor, an SGLT2 inhibitor, a DPP-4 inhibitor, an HMG-CoA reductase inhibitor, a statin, and a combination of any of the foregoing. The present disclosure also provides kits comprising any of the dsRNAs of the disclosure or any of the pharmaceutical compositions of the disclosure, and optionally, instructions for use. In one embodiment, the disclosure provides a kit for performing a method of inhibiting expression of an ACVR1C gene in a cell by contacting a cell with a double stranded RNA agent of the disclosure in an amount effective to inhibit expression of the ACVR1C gene in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to a subject. The present disclosure further provides an RNA-induced silencing complex (RISC) comprising an antisense strand of any of the dsRNA agents of the disclosure. The present disclosure is further illustrated by the following detailed description. Docket No.: 121301-22520 ALN-511-WO BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a graph showing mouse ACVR1C mRNA levels as analyzed by RT-qPCR from gonadal, subcutaneous white and brown adipose tissue (n = 3 per group) 21 days after subcutaneous administration of a single dose of the indicated dsRNA duplex, AD-1735913, at either 0.5 mg/kg, 2 mg/kg or 5 mg/kg. Mouse ACVR1C mRNA levels were calculated relative to control group treated with PBS and normalized to PPIA (peptidyl-prolyl cis-trans isomerase) gene. FIG.2 is a graph showing the effect as measured by RT-qPCR at 21 days after a single, 5 mg/kg, subcutaneous administration of either AD-2410459.1, AD-2410460.1, AD-1735894, AD- 2410461.1, or AD-2410462.1 on ACVR1C mRNA levels in brown and white adipose tissues (gonadal and subcutaneous) in mice. Data is represented as % ACVR1C mRNA remaining relative to PBS control group and normalized to UBC (Ubiquitin C) gene. FIG.3 is a graph showing the ACVR1C % mRNA remaining in NHP relative to average pre- dose at day -15 and day -2 following administration of AD-2640052, AD-2640053, AD-2640060, or PBS. FIG.4 is a graph showing the ACVR1C % mRNA remaining relative to average PBS group at day 85 in NHP adipose tissues following administration of AD-2640052, AD-2640053, AD- 2640060, or PBS. FIG.5 is a graph showing the ALK7 peptide area ratio % in NHP of AD-2640052, AD- 2640053, AD-2640060, AD-2509191, AD-2508176, or PBS groups at day -15, day -2, day 43, and day 57. FIG.6 is a graph showing the ALK7 peptide area ratio % in NHP relative to average pre-dose timepoints at day -15 and day -2 of groups AD-2640052, AD-2640053, AD-2640060, and PBS. FIG.7 is a graph showing ALK7 peptide area ratio % in NHP relative to average PBS of groups AD-2640052, AD-2640053, AD-2640060, and PBS in terminal adipose tissues at day 85. DETAILED DESCRIPTION OF THE DISCLOSURE The present disclosure provides iRNA compositions which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an activin A receptor type 1C (ACVR1C) gene. The gene may be within a cell, such as an adipocyte, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (an ACVR1C gene) in mammals. The iRNAs of the disclosure have been designed to target the human ACVR1C 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 iRNAs of the disclosure improved efficacy, stability, potency, durability, and safety. Accordingly, the present disclosure provides methods for treating and preventing an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome; a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes; a lipid metabolism disorder, e.g., a hyperlipidemia, Docket No.: 121301-22520 ALN-511-WO hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight, using dsRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ACVR1C gene. 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, 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 an ACVR1C gene. In certain embodiments, 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 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. In some embodiments, 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. The use of iRNAs of the disclosure enables the targeted degradation of the ACVR1C 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 ACVR1C gene. Thus, methods and compositions including these iRNAs are useful for treating a subject having an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight, or for treating a subject at risk of developing an ACVR1C-associated disorder. Accordingly, 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 an ACVR1C gene, e.g., an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome; a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes; a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight, using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ACVR1C gene. 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 an ACVR1C gene, e.g., an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome; a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes; a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight. The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of ACVR1C, as well as compositions, uses, and methods for treating Docket No.: 121301-22520 ALN-511-WO subjects that would benefit from inhibition and/or reduction of the expression of ACVR1C, e.g., subjects susceptible to or diagnosed with an ACVR1C-associated disorder, e.g., a metabolic disorder. I. Definitions In order that the present disclosure may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this disclosure. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements. The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to". The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise. For example, “sense strand or antisense strand” is 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%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range. The term “at least”, “no less than”, or “or more” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range. As used herein, “no more than” or “or less” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit. As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method. In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence. In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence. Docket No.: 121301-22520 ALN-511-WO As used herein, “activin A receptor type 1C,” used interchangeably with the terms h57HE*7&i \QRQ\] ^Z M ^c[Q = \QOQ[^Z\ RZ\ ^TQ G;:'q RMXUWc ZR ]USYMWUYS XZWQO_WQ( 57HE*7 TM] intrinsic serine/threonine kinase activities in its cytoplasmic domains, inducing phosphorylation and activation of the SMAD2/3/4 complex, which translocates into the nucleus where it binds SMAD- binding elements (SBE) to regulate (e.g., activate and/or suppress) gene transcription. Expression levels of ACVR1C vary greatly among tissues, but white and brown adipose tissues have the highest level of expression. ACVR1C is also known as “activin receptor-like kinase 7” (ALK-7). The sequence of a human ACVR1C mRNA transcript can be found at, for example, GenBank Accession No. GI: 1519315475 (NM_145259.3, SEQ ID NO:1; reverse complement, SEQ ID NO:2), GI: 1890343165 (NM_001111031.2, SEQ ID NO:3; reverse complement, SEQ ID NO:4), GI: 1676439980 (NM_001111032.2, SEQ ID NO:5, reverse complement SEQ ID NO:6), and GI: 1676318472 (NM_001111033.2, SEQ ID NO:7, reverse complement, SEQ ID NO:8). The sequence of mouse ACVR1C mRNA can be found at, for example, GenBank Accession No. GI: 161333830 (NM_001111030.1, SEQ ID NO:9; reverse complement, SEQ ID NO:10) or GI: 161333829 (NM_001033369.3, SEQ ID NO:11; reverse complement, SEQ ID NO:12). The sequence of rat ACVR1C mRNA can be found at, for example, GenBank Accession No. GI: 1937875934 (NM_139090.2; SEQ ID NO:13; reverse complement, SEQ ID NO:14). The sequence of Macaca mulatta ACVR1C mRNA can be found at, for example, GenBank Accession No. GI: 388454445 (NM_001266690.1; SEQ ID NO:15; reverse complement, SEQ ID NO:16). The sequence of Macaca fascicularis ACVR1C mRNA can be found at, for example, GenBank Accession No. GI: 2161825093 (XM_005573226.3; SEQ ID NO:17; reverse complement, SEQ ID NO:18). The sequence of Macaca fascicularis ACVR1C mRNA can be found at, for example, XM_015432222.1; (SEQ ID NO:19; reverse complement, SEQ ID NO:21); or XM_005573228.2 (SEQ ID NO:21; reverse complement, SEQ ID NO:22); or XM_005573225.2 (SEQ ID NO: 23; reverse complement, SEQ ID NO:24). The sequence of Macaca fascicularis ACVR1C mRNA can be found at, for example, GenBank Accession No. GI: 2161825091 (XM_005573224.3; SEQ ID NO:25; reverse complement, SEQ ID NO:26). The sequence of Macaca fascicularis ACVR1C mRNA can be found at, for example, GenBank Accession No. GI: 2161825094 (XM_005573230.3; SEQ ID NO:27; reverse complement, SEQ ID NO:28). Additional examples of ACVR1C mRNA sequences are readily available through publicly available databases, e.g., GenBank, UniProt, OMIM, the UCSC Genome Browser, and the Macaca genome project web site. Further information on ACVR1C can be found, for example, at www.ncbi.nlm.nih.gov/gene/?term= ACVR1C. The term ACVR1C, as used herein, also refers to variations of the ACVR1C gene including variants provided in the SNP database. Numerous sequence variations within the ACVR1C gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., www.ncbi.nlm.nih.gov/snp/?term= ACVR1C, the entire contents of which is incorporated herein by reference as of the date of filing this application). The entire contents of each of the foregoing Docket No.: 121301-22520 ALN-511-WO GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application. As used herein, “target sequence” or “target nucleic acid” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ACVR1C gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ACVR1C gene. In one embodiment, the target sequence is within the protein coding region of the ACVR1C gene. In another embodiment, the target sequence is within the 3’ UTR of the ACVR1C 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. The target sequence may be from about 19-36 nucleotides in length, e.g., about 19-30 nucleotides in length. For example, 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 nucleotides in length. In certain embodiments, 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. As used herein, the term “strand comprising a sequence” refers to an oligonucleotide 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. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that 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. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the disclosure by a nucleotide containing, for example, inosine. In another example, 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. The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer 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. iRNA directs the sequence-specific degradation of mRNA through a process known as Docket No.: 121301-22520 ALN-511-WO RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of an ACVR1C gene in a cell, e.g., an adipocyte, within a subject, such as a mammalian subject. In one embodiment, an RNAi agent of the disclosure includes a single stranded RNA that interacts with a target RNA sequence, e.g., an ACVR1C mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). 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 (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). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15:188). Thus, in one aspect the disclosure relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the ACVR1C gene. Accordingly, the term “siRNA” is also used herein to refer to an iRNA as described above. In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) that is 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. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894. In certain embodiments, 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”. The term “dsRNA”, refers to a 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., an ACVR1C mRNA sequence. In some embodiments of the disclosure, 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 interference or RNAi. In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide or a modified nucleotide. In addition, as used in this specification, an “iRNA” may include ribonucleotides with chemical modifications; an iRNA may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified Docket No.: 121301-22520 ALN-511-WO internucleotide linkage, or modified nucleobase, or any combination thereof. Thus, the term 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 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. In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide if present within an RNAi agent can be considered to constitute a modified nucleotide. 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, 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. In certain embodiments, 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 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 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. In some embodiments, 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 loop can be 4-8 nucleotides. In certain embodiment, 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. When the two strands are linked to each other at both ends, 5'-end of 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. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleosidic linker, Docket No.: 121301-22520 ALN-511-WO e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker. 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 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. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not be, but can be covalently connected. Where the two strands are connected covalently by means other than 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 structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand comprises a 3’ overhang of at least 1 nucleotide. In another embodiment, at least one 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. In other embodiments, at least one strand of the RNAi agent comprises a 5’ overhang of at least 1 nucleotide. In certain embodiments, 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. In still other embodiments, both the 3’ and the 5’ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide. In certain embodiments, 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., an ACVR1C mRNA sequence, to direct cleavage of the target RNA. In some embodiments, an iRNA of the disclosure is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence, e.g., an ACVR1C mRNA sequence, to direct the cleavage of the target RNA. As used herein, the term “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 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 Docket No.: 121301-22520 ALN-511-WO 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 either an antisense or sense strand of a dsRNA. In one embodiment, 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. In one embodiment, 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. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, 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. In one embodiment, 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. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, 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. In certain embodiments, the 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. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3’ end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5’ end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, 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 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 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. The term “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., an ACVR1C mRNA. As used herein, the term “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., an ACVR1C 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 Docket No.: 121301-22520 ALN-511-WO molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5’- or 3’-end of the iRNA. In some embodiments, a double stranded RNA agent of the disclosure includes a nucleotide mismatch in the antisense strand. In some embodiments, 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 the target mRNA. In some embodiments, 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. In some embodiments, a double stranded RNA agent of the disclosure includes a nucleotide mismatch in the sense strand. In some embodiments, 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. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3’-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3’-terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) is not in the seed region. Thus, an RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, 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 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. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of ACVR1C, 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 an ACVR1C gene is important, especially if the particular region of complementarity in the target gene is known to have polymorphic sequence variation within the population. The term “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. 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. As used herein, the term “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. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on Docket No.: 121301-22520 ALN-511-WO 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. As used herein, and unless otherwise indicated, the term “complementary,” when used to 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. 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, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an 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. However, where a 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 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. However, where 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. For example, a dsRNA comprising one oligonucleotide 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 the above requirements with respect to their ability to hybridize are fulfilled. Such 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 double stranded RNA agent and a target sequence, as will be understood from the context of their use. As used herein, 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 an ALK7 protein). For example, a polynucleotide is complementary to at least a part of an ACVR1C mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ALK7. Docket No.: 121301-22520 ALN-511-WO Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target gene sequence. In some embodiments, 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, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, for ACVR1C, or a fragment of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target ACVR1C 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 sequences in any one of Tables 2-5, 7, 8, 11, and 12, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary. In some embodiments, the sense and antisense strands are selected from any one of duplexes AD-1735894, AD-1735913, AD-2410459, AD-2410460, AD-2410461, and AD-2410462. In some embodiments, the sense and antisense strands are selected from duplex AD-1735894. In some embodiments, the sense and antisense strands are selected from duplex AD-1735913. In some embodiments, the sense and antisense strands are selected from duplex AD-2410459. In some embodiments, the sense and antisense strands are selected from duplex AD-2410460. In some embodiments, the sense and antisense strands are selected from duplex AD-2410461. In some embodiments, the sense and antisense strands are selected from duplex AD-2410462. In one embodiment, 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 ACVR1C 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, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, or a fragment of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary. In some embodiments, 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 ACVR1C 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 2-5, 7, 8, 11, and 12, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12 such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary. Docket No.: 121301-22520 ALN-511-WO In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In one embodiment, 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. In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length. In one embodiment, the sense strand of the iRNA agent is 21-nucleotides in length, and the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single stranded overhangs at the 3'-end. In general, 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. In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide if present within an RNAi agent can be considered to constitute a modified nucleotide. In an aspect of the disclosure, 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 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. For example, the single- 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. 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. Thus, for example, the iRNA may be put into physical contact with the cell by the Docket No.: 121301-22520 ALN-511-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 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. For example, the iRNA may contain or be coupled to a targeting ligand, e.g., GalNAc, that directs the iRNA to a site of interest, e.g., the liver. In other embodiments, the RNAi agent may contain or be coupled to one or more C22 hydrocarbon chains and one or more GalNAc derivatives. In other embodiments, the RNAi agent contains or is coupled to one or more C22 hydrocarbon chains and does not contain or is not coupled to one or more GalNAc derivatives. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject. In certain embodiments, 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 agents or devices. Introducing an iRNA into a cell may be in vitro or in vivo. For example, for in vivo introduction, 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. Further approaches are described herein below or are known in the art. The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a 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. As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a 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. In an embodiment, the subject is a human, such as a human being treated or assessed for a disease or disorder that would benefit from reduction in ACVR1C expression; a human at risk for a disease or disorder that would benefit from reduction in ACVR1C expression; a human having a disease or disorder that would benefit from reduction in ACVR1C expression; or human being treated for a disease or disorder that would benefit from reduction in ACVR1C expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result, such as reducing at least one sign or symptom associated with ACVR1C expression or ALK7 protein production, e.g., an ACVR1C-associated disease, e.g., a metabolic disorder, in a subject. Treatment Docket No.: 121301-22520 ALN-511-WO also includes a reduction of one or more sign or symptoms associated with unwanted ACVR1C expression; diminishing the extent of unwanted ACVR1C activation or stabilization; amelioration or palliation of unwanted ACVR1C 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 ACVR1C in a subject or a disease marker or 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. In certain embodiments, a decrease is at least 20%. In certain embodiments, 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 ACVR1C in a subject is a decrease to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, “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 an ACVR1C-associated disorder towards or to a level in a normal subject not suffering from an ACVR1C-associated disorder. As used herein, if a disease is associated with an elevated value for a symptom, “normal” is considered to be the upper limit of normal. If a disease is associated with a decreased value for a symptom, “normal” is considered to be the lower limit of normal. As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, may be treated or ameliorated by a reduction in expression of an ACVR1C gene or production of an ALK7 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 an ACVR1C- associated disorder, e.g., a metabolic disorder. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention. As used herein, the term “ACVR1C-associated disorder” or “ACVR1C-associated disease” is a disease or disorder that would benefit from reduction in the mRNA expression or activity of ACVR1C. The term “ACVR1C-associated disease,” is a disease or disorder that is caused by, or associated with, ACVR1C mRNA expression or ALK7 protein production. The term “ACVR1C- associated disease” includes a disease, disorder or condition that would benefit from a decrease in ACVR1C mRNA expression or ALK7 protein activity. In some embodiments, the ACVR1C-associated disease is a metabolic disorder. A “metabolic disorder” is a disorder that disrupts normal metabolism, the process of converting food to energy on a cellular level. Metabolic diseases affect the ability of the cell to perform critical biochemical reactions that involve the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). Non-limiting examples of metabolic Docket No.: 121301-22520 ALN-511-WO diseases include disorders of carbohydrates, e.g., diabetes, type I diabetes, type II diabetes, galactosemia, hereditary fructose intolerance, fructose 1,6-diphosphatase deficiency, glycogen storage disorders, congenital disorders of glycosylation, insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism; disorders of amino acid metabolism, e.g., maple syrup urine disease (MSUD), or homocystinuria; disorder of organic acid metabolism, e.g.,methylmalonic aciduria, 3-methylglutaconic aciduria -Barth syndrome, glutaric aciduria or 2-hydroxyglutaric aciduria – D and L forms; disorders of fatty acid beta-oxidation, e.g., medium-chain acyl-CoA dehydrogenase deficiency (MCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHAD), very-long-chain acyl-CoA dehydrogenase deficiency (VLCAD); disorders of lipid metabolism, e.g., GM1 Gangliosidosis, Tay-Sachs Disease, Sandhoff Disease, Fabry Disease, Gaucher Disease, Niemann-Pick Disease, Krabbe Disease, Mucolipidoses, or Mucopolysaccharidoses; disorders of lipid distribution and/or storage, e.g., lipodystrophy, mitochondrial disorders, e.g., mitochondrial cardiomyopathies; Leigh disease; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); myoclonic epilepsy with ragged- red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); Barth syndrome; peroxisomal disorders, e.g., Zellweger Syndrome (cerebrohepatorenal syndrome), X-Linked Adrenoleukodystrophy or Refsum Disease. In one embodiment, a metabolic disorder is metabolic syndrome. The term “metabolic syndrome,” as used herein, is disorder that includes a clustering of components that reflect overnutrition, sedentary lifestyles, genetic factors, increasing age, and resultant excess adiposity. Metabolic syndrome includes the clustering of abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure and is associated with other comorbidities including the prothrombotic state, proinflammatory state, nonalcoholic fatty liver disease, and reproductive disorders. The prevalence of the metabolic syndrome has increased to epidemic proportions not only in the United States and the remainder of the urbanized world but also in developing nations. Metabolic syndrome is associated with an approximate doubling of cardiovascular disease risk and a 5-fold increased risk for incident type 2 diabetes mellitus. Abdominal adiposity (e.g., a large waist circumference (high waist-to-hip ratio)), high blood pressure, insulin resistance and dislipidemia are central to metabolic syndrome and its individual components (e.g., central obesity, fasting blood glucose (FBG)/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension). In one embodiment, a metabolic disorder is a disorder of carbohydrates. In one embodiment, the disorder of carbohydrates is diabetes. As used herein, the term “diabetes” refers to a group of metabolic disorders characterized by high blood sugar (glucose) levels which result from defects in insulin secretion or action, or both. Exemplary symptoms of diabetes may include, frequent urination, feeling more thirsty than usual, blurry vision, slow-healing sores, numb or tingling hands or feet, dry skin, and frequent infections, such as gum, skin and vaginal infections. There are two most common types of diabetes, namely type 1 diabetes and type 2 diabetes, which both result from the body's inability to regulate insulin. Insulin Docket No.: 121301-22520 ALN-511-WO is a hormone released by the pancreas in response to increased levels of blood sugar (glucose) in the blood. The term “type I diabetes,” as used herein, refers to a chronic disease that occurs when the pancreas produces too little insulin to regulate blood sugar levels appropriately. Type I diabetes is also referred to as insulin-dependent diabetes mellitus, IDDM, and juvenile onset diabetes. People with type I diabetes (insulin-dependent diabetes) produce little or no insulin at all. Although about 6 percent of the United States population has some form of diabetes, only about 10 percent of all diabetics have type I disorder. Most people who have type I diabetes develop the disorder before age ,)( Gc[Q * PUMNQ^Q] \Q[\Q]QY^] ^TQ \Q]_W^ ZR M [\ZS\Q]]U`Q M_^ZUXX_YQ PQ]^\_O^UZY ZR ^TQ [MYO\QM^UO q' cells with subsequent insulin deficiency. More than 90 percent of the insulin-producing cells (beta cells) of the pancreas are permanently destroyed. The resulting insulin deficiency is severe, and to survive, a person with type I diabetes must regularly inject insulin. People who have type 1 diabetes may also have nausea, vomiting, or stomach pains. In type II diabetes (also referred to as noninsulin-dependent diabetes mellitus, NDDM), the pancreas continues to manufacture insulin, sometimes even at higher than normal levels. However, the body develops resistance to its effects, resulting in a relative insulin deficiency. Type II diabetes may occur in children and adolescents but usually begins after age 30 and becomes progressively more common with age: about 15 percent of people over age 70 have type II diabetes. Obesity is a risk factor for type II diabetes, and 80 to 90 percent of the people with this disorder are obese. In some embodiments, diabetes includes pre-diabetes. “Pre-diabetes” refers to one or more early diabetic conditions including impaired glucose utilization, abnormal or impaired fasting glucose levels, impaired glucose tolerance, impaired insulin sensitivity and insulin resistance. Prediabetes is a major risk factor for the development of type 2 diabetes mellitus, cardiovascular disease and mortality. In some embodiments, diabetes includes secondary or other specific types of diabetes, e.g., includes monogenic defects of beta cell function, genetic defects of insulin action, exocrine pancreatic disease, endocrinopathies, drug/chemical induced, infectious, and uncommon immune-mediated and genetic syndromes associated with diabetes. Exemplary genetic defects of beta cell function may include, but are not limited to, maturity-onset diabetes of youth 3 (MODY3) (Chromosome 12, HNF- *l$& AC8J* #7T\ZXZ]ZXQ +)& <B:'-l$& AC8J+ #7T\ZXZ]ZXQ 0& SW_OZVUYM]Q$& Z^TQ\ `Q\c \M\Q forms of MODY (e.g., MODY4: Chromosome 13, insulin promoter factor-1; MODY6: Chromosome 2, NeuroD1; MODY7: Chromosome 9, carboxyl ester lipase); transient neonatal diabetes (most commonly ZAC/HYAMI imprinting defect on 6q24), or permanent neonatal diabetes (most commonly KCNJ11 gene encoding Kir6.2 subunit of beta cell KATP channel). Exemplary genetic defects in insulin action may include, but are not limited to, Type A insulin resistance, Leprechaunism, Rabson-Mendenhall syndrome, lipoatrophic diabetes, or mutant insulins. Exemplary diseases of the exocrine pancreas may include, but are not limited to, pancreatitis, trauma/pancreatectomy, neoplasia, Cystic fibrosis, hemochromatosis, or fibrocalculous pancreatopathy. Exemplary endocrinopathies may include, but are not limited to, acromegaly, Docket No.: 121301-22520 ALN-511-WO Cushing’s syndrome, glucagonoma, pheochromocytoma, somatostatinoma, aldosteronoma, or hyperthyroidism. Exemplary drug/chemical induced diabetes may include, but are not limited to, `MOZ\& [QY^MXUPUYQ& YUOZ^UYUO MOUP& SW_OZOZ\^UOZUP]& ^Tc\ZUP TZ\XZYQ& PUMdZbUPQ& q'5P\QYQ\SUO MSZYU]^]& ^TUMdUPQ]& PUWMY^UY& Z\ r'=Y^Q\RQ\ZY' UYP_OQP PUMNQ^Q]( 9bQX[WM\c UYRQO^UZY] M]]ZOUM^QP aU^T diabetes may include, but are not limited to, congenital rubella, Cytomegalovirus, or mumps. Exemplary uncommon immune-mediated and genetic syndromes associated with diabetes may include, but are not limited to, “Stiff-man” syndrome, anti-insulin receptor antibodies, Down syndrome, Klinefelter syndrome, Turner syndrome, Wolfram syndrome, Friedreich ataxia, Huntington chorea, Laurence-Moon-Biedl syndrome,Myotonic dystrophy, Porphyria, or Prader-Willi syndrome.Diabetes can be diagnosed by the administration of a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Primary examples of these categories include, autoimmune diabetes mellitus, non-insulin-dependent diabetes mellitus (type 1 NDDM), insulin- dependent diabetes mellitus (type 2 IDDM), non-autoimmune diabetes mellitus, non-insulin- dependent diabetes mellitus (type 2 NIDDM), and maturity-onset diabetes of the young (MODY). A further category, often referred to as secondary, refers to diabetes brought about by some identifiable condition which causes or allows a diabetic syndrome to develop. Examples of secondary categories include, diabetes caused by pancreatic disease, hormonal abnormalities, drug- or chemical-induced diabetes, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes. (see e.g., Harrison's (1996) 14th ed., New York, McGraw- Hill). In one embodiment, a metabolic disorder is a lipid metabolism disorder. As used herein, a “lipid metabolism disorder” or "disorder of lipid metabolism" refers to any disorder associated with or caused by a disturbance in lipid metabolism. This term also includes any disorder, disease or condition that can lead to hyperlipidemia, or condition characterized by abnormal elevation of levels of any or all lipids and/or lipoproteins in the blood. This term refers to an inherited disorder, such as familial hypertriglyceridemia, familial partial lipodystrophy type (FPLD) (e.g., familial partial lipodystrophy type 1 (FPLD1) or Köbberling type, FPLD 2 or Dunnigan type, FPLD 3 or PPARG- related FPLD, FPLD 4 or PLIN1-related FPLD, FPLD 5 or CIDEC-related FPLD, FPLD 6 or LIPE- related FPLD, CAV1-related FPLD, ADRA2A-related FPLD, AKT2-related FPLD, Progeroid syndromes, MAD type A, Werner syndrome, MDPL syndrome, Complex genetic syndromes with partial lipodystrophy, Bloom syndrome, SHORT syndrome, Autoinflammatory syndromes, or CANDLE syndrome), or an induced or acquired disorder, such as a disorder induced or acquired as a result of a disease, disorder or condition (e.g., renal failure), a diet, or intake of certain drugs (e.g., as a result of highly active antiretroviral therapy (HAART) used for treating, e.g., AIDS or HIV). This term also refers to a disorder of fat distribution/storage, e.g., lipodystrophy. Additional examples of disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, PU_\Q^UO'UYP_OQP Tc[Q\^\USWcOQ\UPQXUM& MWOZTZW'UYP_OQP Tc[Q\^\USWcOQ\UPQXUM& q'MP\QYQ\SUO NWZOVUYS agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, post-menopausal lipid Docket No.: 121301-22520 ALN-511-WO metabolism disorder, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrom, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, lipodystrophy, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), hyperlipidemia with heterogeneous LPL deficiency, hyperlipidemia with high LDL and heterogeneous LPL deficiency, fatty liver disease, or non-alcoholic stetohepatitis (NASH), or skin disorders associated with disruptions in lipid metabolism (e.g., UV-induced disruptions in lipid metabolism and associated skin disorders, sensitive skin, or psoriasis). Cardiovascular diseases are also considered “metabolic disorders”, as defined herein. These diseases may include coronary artery disease (also called ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular diseases, and stroke. Kidney diseases are also considered “metabolic disorders”, as defined herein. Such diseases may include chronic kidney disease, diabetic nephrophathy, diabetic kidney disease, or gout. Disorders related to body weight are also considered “metabolic disorders”, as defined herein. Such disorders may include obesity, hypo-metabolic states, hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight regain following weight loss. Blood sugar disorders are further considered “metabolic disorders”, as defined herein. Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome related to insulin resistance. Other exemplary disorders of metabolic disorders may also include renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease. In one embodiment, a metabolic disorder is primary hypertension. “Primary hypertension” is a result of environmental or genetic causes (e.g., a result of no obvious underlying medical cause). In one embodiment, a metabolic disorder disorder is secondary hypertension. “Secondary hypertension” has an identifiable underlying disorder which can be of multiple etiologies, including renal, vascular, and endocrine causes, e.g., renal parenchymal disease (e.g., polycystic kidneys, glomerular or interstitial disease), renal vascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disorders (e.g., adrenocorticosteroid or mineralocorticoid excess, pheochromocytoma, hyperthyroidism or hypothyroidism, growth hormone excess, hyperparathyroidism), coarctation of the aorta, or oral contraceptive use. In one embodiment, a metabolic disorder is resistant hypertension. “Resistant hypertension” is blood pressure that remains above goal (e.g., above 130 mm Hg systolic or above 90 diastolic) in spite of concurrent use of three antihypertensive agents of different classes, one of which is a thiazide diuretic. Subjects whose blood pressure is controlled with four or more medications are also considered to have resistant hypertension. Docket No.: 121301-22520 ALN-511-WO Additional diseases or conditions related to metabolic disorders that would be apparent to the skilled artisan and are within the scope of this disclosure. The symptoms for an ACVR1C-associated disease, e.g., a metabolic disorder, include, for example, insulin resistance, lack of ability to regulate blood sugar, a loss of fat-free or lean muscle mass, an excess of fat mass, a lower metabolic rate, weight gain, and/or increase in body mass index. Further details regarding signs and symptoms of the various diseases or conditions are provided herein and are well known in the art. "Therapeutically effective amount," as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having an ACVR1C-associated disorder, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease 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 RNAi agent that, when administered to a subject having an ACVR1C-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 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 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. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, 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. The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically- acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the Docket No.: 121301-22520 ALN-511-WO subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection. The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, 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 adipose (e.g., certain segments of adipose tissue or certain types of cells in the adipose, such as, e.g., adipocytes). In some embodiments, samples may be derived from subcutaneous white adipose tissue (scWAT) (e.g., abdominal or inguinal), visceral white adipose tissue (VWAT) (e.g., mesenteric, gonadal, perirenal), or brown adipose tissue (BAT) (e.g., interscapular, periaortic). In some embodiments, a “sample derived from a subject” refers to urine obtained from the subject. A “sample derived from a subject” can refer to blood or blood derived serum or plasma from the subject. II. iRNAs of the Disclosure The present disclosure provides iRNAs which inhibit the expression of an ACVR1C gene. In certain embodiments, the iRNA includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ACVR1C gene in a cell (e.g., an adipocyte), such as a cell within a subject, e.g., a mammal, such as a human susceptible to developing an ACVR1C-associated disease, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight. 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 an ACVR1C 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). Upon contact with a cell expressing the ACVR1C gene, the iRNA inhibits the expression of the ACVR1C 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 or LCMS, using, for example, western blotting or flow cytometric techniques. In certain embodiments, inhibition of expression is determined by the qPCR method provided in the examples herein with the siRNA at, e.g., a 10 nM concentration, in an appropriate organism cell line provided therein. In certain embodiments, 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. Docket No.: 121301-22520 ALN-511-WO 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 strand) 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 an ACVR1C gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self- complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. Generally, 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, 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. In certain embodiments, 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- 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. Similarly, 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- 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. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure. In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length. In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. In general, 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 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). One of skill in the art will also recognize that 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, Docket No.: 121301-22520 ALN-511-WO 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. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target ACVR1C 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 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. 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. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single- stranded oligonucleotides of the disclosure can be prepared using solution-phase or solid-phase organic synthesis or both. In an aspect, 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 provided in any one of Tables 2-5, 7, 8, 11, and 12, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 2-5, 7, 8, 11, and 12. In this aspect, 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. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 2-5, 7, 8, 11, and 12, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-5, 7, 8, 11, and 12. In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide. In some embodiments, the sense or antisense strands are selected from the sense or antisense strand of any one of duplexes AD-1735894, AD-1735913, AD-2410459, AD-2410460, AD-2410461, Docket No.: 121301-22520 ALN-511-WO AD-2410462, AD-2509512, AD-2640052, AD-2640053, AD-2640060, AD-2509191, AD-2509494, AD-2509477, and AD-2508176. It will be understood that, although the sequences in, for example, Table 2, are not described as modified or conjugated sequences, 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 2-5, 7, 8, 11, and 12 that is un-modified, un-conjugated, or modified or conjugated differently than described therein. In other words, the disclosure encompasses dsRNA of Tables 2-5, 7, 8, 11, and 12 which are un- modified, un-conjugated, modified, or conjugated, as described herein. The skilled person is well aware that 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 (Elbashir et al., EMBO 2001, 20:6877-6888). However, 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). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Tables 2-5, 7, 8, 11, and 12. dsRNAs described herein can include at 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 2-5, 7, 8, 11, and 12 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, 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 2-5, 7, 8, 11, and 12, and differing in their ability to inhibit the expression of an ACVR1C 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. In addition, the RNAs provided in Tables 2-5, 7, 8, 11, and 12 identify a site(s) in an ACVR1C transcript that is susceptible to RISC-mediated cleavage. As such, the present disclosure further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an 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 2-5, 7, 8, 11, and 12 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ACVR1C gene. III. Modifications for the RNAi Agents of the Disclosure In certain embodiments, the RNA of the iRNA of the disclosure, e.g., a dsRNA, is un- modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of an iRNA of the disclosure, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the disclosure, substantially all of the nucleotides of an iRNA of the disclosure are modified. In other embodiments of the disclosure, all of the nucleotides of an iRNA or substantially all of the Docket No.: 121301-22520 ALN-511-WO nucleotides of an iRNA are modified, i.e., not more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in a strand of the iRNA. In some embodiments, the dsRNA agents of the disclosure comprise at least one nucleic acid modification described herein. For example, at least one modification selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combinations thereof. Without limitations, such a modification can be present anywhere in the dsRNA agent of the disclosure. For example, the modification can be present in one of the RNA molecules. In one embodiment, the dsRNA agents of the disclosure comprise one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand and do not comprise additional chemical modifications known in the art and described herein, in the remaining positions of the sense and anti-sense strands. In some embodiments, the dsRNA agents of the disclosure comprise one or more C22 hydrocarbon chains conjugated to one or more internal positions on at least one strand, and comprise at least one additional nucleic acid modification described herein. For example, at least one modification selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combinations thereof. Without limitations, such a modification can be present anywhere in the dsRNA agent of the disclosure. For example, the modification can be present in one of the RNA molecules. 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 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; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified RNAi agent will have a phosphorus atom in its internucleoside backbone. A. Nucleobase Modifications The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. For those Y_OWQZ]UPQ] ^TM^ UYOW_PQ M [QY^ZR_\MYZ]cW ]_SM\& M [TZ][TM^Q S\Z_[ OMY NQ WUYVQP ^Z ^TQ +o& ,o Z\ .o hydroxyl moiety of the sugar. In forming oligonucleotides, those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, Docket No.: 121301-22520 ALN-511-WO the phosphate groups are commonly referred to as forming the internucleoside backbone of the ZWUSZY_OWQZ^UPQ( GTQ YM^_\MWWc ZOO_\\UYS WUYVMSQ Z\ NMOVNZYQ ZR EB5 MYP ZR 8B5 U] M ,o ^Z .o phosphodiester linkage. In addition to “unmodified” or “natural” nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein. The unmodified or natural nucleobases can be modified or replaced to provide iRNAs having improved properties. For example, nuclease resistant oligonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and “universal bases” can be employed. When a natural base is replaced by a non-natural and/or universal base, the nucleotide is said to comprise a modified nucleobase and/or a nucleobase modification herein. Modified nucleobase and/or nucleobase modifications also include natural, non-natural and universal bases, which comprise conjugated moieties, e.g. a ligand described herein. Conjugate moieties for conjugation with nucleobases include cationic amino groups which can be conjugated to the nucleobase via an appropriate alkyl, alkenyl or a linker with an amide linkage. An oligomeric compound described herein can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2- (aminoalkyll)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine, 8-(thioalkyl)adenine, 8- (thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine, N6, N6-(dimethyl)adenine, 2- (alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8- (amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino- 3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)- 2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5- (alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5- (dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil- Docket No.: 121301-22520 ALN-511-WO 5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4- (dithio)psuedouracil,5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5- (methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5- (alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4- (dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)- pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4- (dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino- carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1- yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3- (aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2- (thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1- (aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)- 2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7- (guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidiniumalkylhydroxy)-1- (aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)- phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5- (triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5- (methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7- (aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7- (propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6- (dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6- (azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6- (diamino)purine, 5-substituted pyrimidines, N2-substituted purines, N6-substituted purines, O6- substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo- pyrimidin-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-substituted-6- phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-(aminoalkylhydroxy)- 6- phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho--(aminoalkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2- on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivatives thereof. Alternatively, substituted or modified analogs of any of the above bases and “universal bases” can be employed. Docket No.: 121301-22520 ALN-511-WO As used herein, a universal nucleobase is any nucleobase that can base pair with all of the four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the iRNA duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro- 6-methylbenzimidazle, 4-methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9- methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7- azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447). Further nucleobases include those disclosed in U.S. Pat. No.3,687,808; those disclosed in International Application No. PCT/US09/038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; those disclosed by English et al., Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P.Ed. Wiley-VCH, 2008; and those disclosed by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993. Contents of all of the above are herein incorporated by reference. In certain embodiments, a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5-methyl cytosine, or a G- clamp. In certain embodiments, nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art. B. Sugar Modifications DsRNA agent of the disclosures provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomer, including a nucleoside or nucleotide, having a modified sugar moiety. For example, the furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non- geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid. In certain embodiments, oligomeric compounds comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers that are LNA. In some embodiments of a locked nucleic acid, the 2^ position of furnaosyl is connected to the 4’ position by a linker selected independently from –[C(R1)(R2)]n–, –[C(R1)(R2)]n–O–, – [C(R1)(R2)]n-N(R1)–, –[C(R1)(R2)]n-N(R1)–O-, —[C(R1R2)]n-O-N(R1)—, –C(R1)=C(R2)–O–, – 7#E*$4Bf& f7#E*$4BfC'& g7#nBE*$'& g7#nBE*$'C'& g7#nC$g& g7#nC$Cg& g7#nF$g& g7#nF$Cg& g7#nF$Fg& gCg& gFU#E*$+'& gF#nC$x- and —N(R1)-; wherein: x is 0, 1, or 2; Docket No.: 121301-22520 ALN-511-WO n is 1, 2, 3, or 4; each R1 and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, TMWZSQY& C>*& B>*>+& F>*& B,& 7CC>*& MOcW #7#nC$g<$& ]_N]^U^_^QP MOcW& 7B& ]_WRZYcW #F#nC$+'>*$& Z\ ]_WRZbcW #F#nC$'>*$3 MYP each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted 7.'7+) M\cW& MOcW #7#nC$g<$& ]_N]^U^_^QP MOcW& M TQ^Q\ZOcOWQ \MPUOMW& M ]_N]^U^_^QP TQ^Q\ZOcOWQ radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group. In some embodiments, each of the linkers of the LNA compounds is, independently, — [C(R1)(R2)]n-, —[C(R1)(R2)]n-O—, —C(R1R2)-N(R1)-O— or —C(R1R2)-O—N(R1)-. In another QXNZPUXQY^& QMOT ZR ]MUP WUYVQ\] U]& UYPQ[QYPQY^Wc& -o'7<2'+o& -o'#7<2)2'+o& -o'#7<2)3'+o& -o'7<2'C'+o& -o'#7<2)2'C'+o& -o'7<2'CgB#E*$'+o MYP -o'7<2'B#E*$'C'+o' aTQ\QUY QMOT E* U]& UYPQ[QYPQY^Wc& <& M protecting group or C1-C12 alkyl. Certain LNA's have been prepared and disclosed in the patent literature as well as in scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94/14226; WO 2005/021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Examples of issued US patents and published applications that disclose LNA s include, for example, U.S. Pat. Nos.7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos.2004-0171570; 2004-0219565; 2004- 0014959; 2003-0207841; 2004-0143114; and 20030082807. 5W]Z [\Z`UPQP TQ\QUY M\Q @B5] UY aTUOT ^TQ +o'TcP\ZbcW S\Z_[ ZR ^TQ \UNZ]cW ]_SM\ \UYS U] WUYVQP ^Z ^TQ -o OM\NZY M^ZX ZR ^TQ ]_SM\ \UYS ^TQ\QNc RZ\XUYS M XQ^TcWQYQZbc #-o'7<2'C'+o$ WUYVMSQ to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 81-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos.6,268,490 and 6,670,461). The linkage can be a methylene (—CH2-) S\Z_[ N\UPSUYS ^TQ +o ZbcSQY M^ZX MYP ^TQ -o OM\NZY M^ZX& RZ\ aTUOT ^TQ ^Q\X XQ^TcWQYQZbc #-o'7<2- C'+o$ @B5 U] _]QP RZ\ ^TQ NUOcOWUO XZUQ^c3 UY ^TQ OM]Q ZR MY Q^TcWQYQ S\Z_[ UY ^TU] [Z]U^UZY& ^TQ ^Q\X Q^TcWQYQZbc #-o'7<2CH2'C'+o$ @B5 U] _]QP #FUYST et al., Chem. Commun., 1998, 4, 455-456: Morita et al.& 6UZZ\SMYUO AQPUOUYMW 7TQXU]^\c& +)),& **& ++**'+++/$( AQ^TcWQYQZbc #-o'7<2'C'+o$ @B5 MYP other bicyclic sugar analogs display very high duplex thermal stabilities with complementary DNA MYP EB5 #GX4%, ^Z %*)e 7($& ]^MNUWU^c ^ZaM\P] ,o'QbZY_OWQZWc^UO PQS\MPM^UZY MYP SZZP ]ZW_NUWU^c properties. Potent and nontoxic antisense oligonucleotides comprising BNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638). 5Y U]ZXQ\ ZR XQ^TcWQYQZbc #-o'7<2'C'+o$ @B5 ^TM^ TM] MW]Z NQQY PU]O_]]QP U] MW[TM'@' XQ^TcWQYQZbc #-o'7<2'C'+o$ @B5 aTUOT TM] NQQY ]TZaY ^Z TM`Q ]_[Q\UZ\ ]^MNUWU^c MSMUY]^ M ,o' Docket No.: 121301-22520 ALN-511-WO QbZY_OWQM]Q( GTQ MW[TM'@'XQ^TcWQYQZbc #-o'7<2'C'+o$ @B5"] aQ\Q UYOZ\[Z\M^QP UY^Z MY^U]QY]Q gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). GTQ ]cY^TQ]U] MYP [\Q[M\M^UZY ZR ^TQ XQ^TcWQYQZbc #-o'7<2'C'+o$ @B5 XZYZXQ\] MPQYUYQ& cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607- 3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226. 5YMWZS] ZR XQ^TcWQYQZbc #-o'7<2'C'+o$ @B5& [TZ][TZ\Z^TUZM^Q'XQ^TcWQYQZbc #-o'7<2'C'+o$ @B5 MYP +o'^TUZ'@B5]& TM`Q MW]Z NQQY [\Q[M\QP #?_XM\ et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). :_\^TQ\XZ\Q& ]cY^TQ]U] ZR +o'MXUYZ'@B5& M YZ`QW OZXRZ\XM^UZYMWWc \Q]^\UO^QP TUST'MRRUYU^c oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035- *)),2$( =Y MPPU^UZY& +o'5XUYZ' MYP +o'XQ^TcWMXUYZ'@B5"] TM`Q NQQY [\Q[M\QP MYP ^TQ ^TQ\XMW stability of their duplexes with complementary RNA and DNA strands has been previously reported. Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance. A representative list of modified sugars includes but is not limited to bicyclic modified sugars, including methyleneoxy #-o'7<2'C'+o$ @B5 MYP Q^TcWQYQZbc #-o'#7<2)2'C'+o N\UPSQ$ 9B53 ]_N]^U^_^QP ]_SM\]& Q][QOUMWWc +o' ]_N]^U^_^QP ]_SM\] TM`UYS M +o':& +o'C7<3 Z\ M +o'C#7<2)2-OCH3 ]_N]^U^_QY^ S\Z_[3 MYP -o'^TUZ modified sugars. Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art. Examples of “oxy”-2^ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR, n =1-50; “locked” nucleic acids (LNA) in which the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2)nAMINE (n = 1-10, AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, ethylene diamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2. “Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the single-strand overhangs); halo (e.g., fluoro); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl and alkynyl, which can be optionally substituted with e.g., an amino functionality. Other suitable 2’-modifications, e.g., modified MOE, are described in U.S. Patent Application Publication No.20130130378, contents of which are herein incorporated by reference. Docket No.: 121301-22520 ALN-511-WO A modification at the 2’ position can be present in the arabinose configuration The term “arabinose configuration” refers to the placement of a substituent on the C2’ of ribose in the same configuration as the 2’-OH is in the arabinose. The sugar can comprise two different modifications at the same carbon in the sugar, e.g., gem modification. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, an oligomeric compound can include one or more monomers containing e.g., arabinose, as the sugar. The monomer can have an alpha linkage at the 1’ position on the sugar, e.g., alpha-nucleosides. The monomer can also have the opposite configuration at the 4’-position, e.g., C5’ and H4’ or substituents replacing them are interchanged with each other. When the C5’ and H4’ or substituents replacing them are interchanged with each other, the sugar is said to be modified at the 4’ position. DsRNA agent of the disclosures disclosed herein can also include abasic sugars, i.e., a sugar which lack a nucleobase at C-1^ or has other chemical groups in place of a nucleobase at C1’. See for example U.S. Pat. No. 5,998,203, content of which is herein incorporated in its entirety. These abasic sugars can also be further containing modifications at one or more of the constituent sugar atoms. DsRNA agent of the disclosures can also contain one or more sugars that are the L isomer, e.g. L- nucleosides. Modification to the sugar group can also include replacement of the 4’-O with a sulfur, optionally substituted nitrogen or CH2 group. In some embodiments, linkage between C1’ and Y_OWQZNM]Q U] UY l OZYRUS_\M^UZY( Sugar modifications can also include acyclic nucleotides, wherein a C-C bonds between ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, C1’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are or in combination absent from the nucleotide. In some embodiments, nucleotide
Figure imgf000046_0001
Figure imgf000046_0002
B is a modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). =Y ]ZXQ QXNZPUXQY^]& ]_SM\ XZPURUOM^UZY] M\Q ]QWQO^QP R\ZX ^TQ S\Z_[ OZY]U]^UYS ZR +j'<& +o' O'AQ #+o'O'XQ^TcW$& +o'O'AC9 #+o'O'XQ^TZbcQ^TcW$& +j':& +o'O'K+'#XQ^TcWMXUYZ$'+'ZbZQ^TcWL #+o' O-NMA), 2’-S-methyl, 2’-O-CH2-(4’-C) (LNA), 2’-O-CH2CH2-(4’-C) (ENA), 2'-O-aminopropyl (2'- O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O- Docket No.: 121301-22520 ALN-511-WO dimethylaminoethyloxyethyl (2'-O-DMAEOE) and gem 2’-OMe/2’F with 2’-O-Me in the arabinose configuration. It is to be understood that when a particular nucleotide is linked through its 2’-position to the next nucleotide, the sugar modifications described herein can be placed at the 3’-position of the sugar for that particular nucleotide, e.g., the nucleotide that is linked through its 2’ -position. A modification at the 3’ position can be present in the xylose configuration The term “xylose configuration” refers to the placement of a substituent on the C3’ of ribose in the same configuration as the 3’-OH is in the xylose sugar. The hydrogen attached to C4’ and/or C1’ can be replaced by a straight- or branched- optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein backbone of the alkyl, alkenyl and alkynyl can contain one or more of O, S, S(O), SO2, N(R’), C(O), N(R’)C(O)O, OC(O)N(R’), CH(Z’), phosphorous containing linkage, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic or optionally substituted cycloalkyl, where R’ is or substituted Z’ is selected from the group consisting
Figure imgf000047_0001
CONR21R31, CON(H)NR21R31, ONR21R31, CON(H)N=CR41R51, N(R21)C(=NR31)NR21R31, N(R21)C(O)NR21R31, N(R21)C(S)NR21R31, OC(O)NR21R31, SC(O)NR21R31, N(R21)C(S)OR11, N(R21)C(O)OR11, N(R21)C(O)SR11, N(R21)N=CR41R51, ON=CR41R51, SO2R11, SOR11, SR11, and substituted or unsubstituted heterocyclic; R21 and R31 for each occurrence are independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR11, COR11, CO2R11, or NR11R11’; or R21 and R31, taken together with the atoms to which they are attached, form a heterocyclic ring; R41 and R51 for each occurrence are independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR11, COR11, or CO2R11, or NR11R11’; and R11 and R11’ are independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to the C4’ of the 5’ terminal nucleotide is replaced. In some embodiments, C4’ and C5’ together form an optionally substituted heterocyclic, preferably comprising at least one -PX(Y)-, wherein X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino, where M is independently for each occurrence an alki metal or transition metal with an overall charge of +1; and Y is O, S, or NR’, where R’ is hydrogen, optionally substituted aliphatic. Preferably this modification is at the 5 terminal of the iRNA. In certain embodiments, LNA's include nucleoside having the formula:
Figure imgf000047_0002
wherein: Docket No.: 121301-22520 ALN-511-WO Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2- C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide. In some embodiments, each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, TcP\ZbcW& C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ MYP 7B& aTQ\QUY QMOT J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In certain such embodiments, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, F>*& B,& C7#nI$>*& MYP B>,7#nI$B>*>+& aTQ\QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY^Wc& <& 71-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJ1. In certain embodiments, the Z group is C1-C6 alkyl substituted with one or more Xx, wherein QMOT Ib U] UYPQ[QYPQY^Wc C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z\ 7B3 wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, the Z group is C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—), substituted alkoxy or azido. In certain embodiments, the Z group is —CH2Xx, wherein Xx is OJ1, NJ1J2, SJ1, N3, C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z\ 7B3 aTQ\QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY^Wc& < or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, the Z group is —CH2Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido. In certain such embodiments, the Z is in the -
Figure imgf000048_0001
In certain such embodiments, the Z is in the -
Figure imgf000048_0002
In certain embodiments, each T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t- butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9- (p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is T1 U] -&-o'PUXQ^TZbc^\U^cW( Docket No.: 121301-22520 ALN-511-WO In certain embodiments, T2 is a reactive phosphorus group wherein preferred reactive phosphorus groups include diisopropylcyanoethoxy phosphoramidite and H-phosphonate. In certain embodiments T1 U] -&-o'PUXQ^TZbc^\U^cW MYP G2 is diisopropylcyanoethoxy phosphoramidite. In certain embodiments, the compounds of the disclosure comprise at least one monomer of the formula:
Figure imgf000049_0001
or of the formula:
Figure imgf000049_0002
or of the formula:
Figure imgf000049_0003
T3 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; T4 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; and Z is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2- C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide. In some embodiments, each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, TcP\ZbcW& C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ MYP 7B& aTQ\QUY QMOT J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In some embodiments, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, Docket No.: 121301-22520 ALN-511-WO F>*& B,& C7#nI$>*& MYP B>,7#nI$B>*>+& aTQ\QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY^Wc& < Z\ 71-C6 alkyl, and X is O or NJ1. In certain such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, each Z is, independently, C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, at least one Z is C1-C6 alkyl. In certain embodiments, each Z is, independently, C1-C6 alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C1-C6 alkyl. In certain embodiments, each Z is, independently, substituted C1-C6 alkyl. In certain embodiments, at least one Z is substituted methyl. In certain embodiments, each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl. In certain embodiments, at least one substituent group is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In another embodiment, each substituent group is, independently, C1-C6 alkoxy (e.g., each Z is, independently, C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In certain embodiments, at least one C1-C6 alkoxy substituent group is CH3O— (e.g., at least one Z is CH3OCH2-). In another embodiment, each C1-C6 alkoxy substituent group is CH3O— (e.g., each Z is CH3OCH2-). In certain embodiments, at least one substituent group is halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogen). In certain embodiments, each substituent group is, independently, halogen (e.g., each Z is, independently, C1-C6 alkyl substituted with one or more halogen). In certain embodiments, at least one halogen substituent group is fluoro (e.g., at least one Z is CH2FCH2-, CHF2CH2- or CF3CH2-). In certain embodiments, each halo substituent group is fluoro (e.g., each Z is, independently, CH2FCH2-, CHF2CH2- or CF3CH2-). In certain embodiments, at least one substituent group is hydroxyl (e.g., at least one Z is C1- C6 alkyl substituted with one or more hydroxyl). In certain embodiments, each substituent group is, independently, hydroxyl (e.g., each Z is, independently, C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, at least one Z is HOCH2-. In another embodiment, each Z is HOCH2-. In certain embodiments, at least one Z is CH3-, CH3CH2-, CH2OCH3-, CH2F— or HOCH2-. In certain embodiments, each Z is, independently, CH3-, CH3CH2-, CH2OCH3-, CH2F— or HOCH2-. In certain embodiments, at least one Z group is C1-C6 alkyl substituted with one or more Xx, aTQ\QUY QMOT Ib U]& UYPQ[QYPQY^Wc& C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z\ 7B3 aTQ\QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY^Wc& < Z\ 71-C6 alkyl, and X is O, S or NJ1. In another embodiment, at least one Z group is C1-C6 alkyl substituted with one or more Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido. In certain embodiments, each Z group is, independently, C1-C6 alkyl substituted with one or XZ\Q Ib& aTQ\QUY QMOT Ib U] UYPQ[QYPQY^Wc C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z\ 7B3 aTQ\QUY QMOT >*& >+ MYP >, U]& UYPQ[QYPQY^Wc& < Z\ 71-C6 alkyl, and X is O, S Docket No.: 121301-22520 ALN-511-WO or NJ1. In another embodiment, each Z group is, independently, C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido. In certain embodiments, at least one Z group is —CH2Xx, wherein Xx is OJ1, NJ1J2, SJ1, B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z\ 7B3 aTQ\QUY QMOT >*& >+ MYP >, U]& independently, H or C1-C6 alkyl, and X is O, S or NJ1 In certain embodiments, at least one Z group is —CH2Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido. In certain embodiments, each Z group is, independently, —CH2Xx, wherein each Xx is, UYPQ[QYPQY^Wc& C>*& B>*>+& F>*& B,& C7#nI$>*& C7#nI$B>*>+& B>,7#nI$B>*>+ Z\ 7B3 aTQ\QUY each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, each Z group is, independently, —CH2Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido. In certain embodiments, at least one Z is CH3-. In another embodiment, each Z is, CH3-. In certain embodiments, the Z group of at least one monomer is in the (R)— configuration represented by the formula:
Figure imgf000051_0001
or the formula:
Figure imgf000051_0002
or the formula:
Figure imgf000051_0003
IN certain embodiments, the Z group of each monomer of the formula is in the (R)— configuration. In certain embodiments, the Z group of at least one monomer is in the (S)— configuration represented by the formula:
Figure imgf000051_0004
or the formula: Docket No.: 121301-22520 ALN-511-WO
Figure imgf000052_0001
or the formula:
Figure imgf000052_0002
In certain embodiments, the Z group of each monomer of the formula is in the (S)— configuration. In certain embodiments, T3 is H or a hydroxyl protecting group. In certain embodiments, T4 is H or a hydroxyl protecting group. In a further embodiment T3 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit. In certain embodiments, T4 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit. In certain embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide. In certain embodiments, T4 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide. In certain embodiments, T3 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, T4 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, at least one of T3 and T4 comprises an internucleoside linking group selected from phosphodiester or phosphorothioate. In certain embodiments, dsRNA agent of the disclosure comprise at least one region of at least two contiguous monomers of the formula:
Figure imgf000052_0003
or of the formula:
Figure imgf000052_0004
or of the formula:
Figure imgf000052_0005
=Y OQ\^MUY ]_OT QXNZPUXQY^]& @B5] UYOW_PQ& N_^ M\Q YZ^ WUXU^QP ^Z& #5$ l'@'AQ^TcWQYQZbc #-o'7<2'C'+o$ @B5& #6$ q'8'AQ^TcWQYQZbc #-o'7<2'C'+o$ @B5& #7$ 9^TcWQYQZbc #-o'#7<2)2'C'+o$ Docket No.: 121301-22520 ALN-511-WO @B5& #8$ 5XUYZZbc #-o'7<2'CgB#E$'+o$ @B5 MYP #9$ CbcMXUYZ #-o'7<2'B#E$gC'+o$ @B5& M] depicted below:
Figure imgf000053_0001
In certain embodiments, the dsRNA agent of the disclosure comprises at least two regions of at least two contiguous monomers of the above formula. In certain embodiments, the dsRNA agent of the disclosure comprises a gapped motif. In certain embodiments, the dsRNA agent of the disclosure OZX[\U]Q] M^ WQM]^ ZYQ \QSUZY ZR R\ZX MNZ_^ 1 ^Z MNZ_^ *- OZY^US_Z_] q'8'+o'PQZbc\UNZR_\MYZ]cW nucleosides. In certain embodiments, the dsRNA agent of the disclosure comprises at least one region ZR R\ZX MNZ_^ 2 ^Z MNZ_^ *+ OZY^US_Z_] q'8'+o'PQZbc\UNZR_\MYZ]cW Y_OWQZ]UPQ]( In certain embodiments, the dsRNA agent of the disclosure comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) comprises at least one (S)-cEt monomer of the formula: Docket No.: 121301-22520 ALN-511-WO
Figure imgf000054_0001
, wherein Bx is heterocyclic base moiety. In certain embodiments, monomers include sugar mimetics. In certain such embodiments, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. Representative examples of a sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of a mimetic for a sugar-internucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art. C. Intersugar Linkage Modifications Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming an oligomeric compound, e.g., an oligonucleotide. Such linking groups are also referred to as intersugar linkage. The two main classes of linking groups are defined by the presence or absence of a phosphorus atom. EQ[\Q]QY^M^U`Q [TZ][TZ\_] OZY^MUYUYS WUYVMSQ] UYOW_PQ& N_^ M\Q YZ^ WUXU^QP ^Z& [TZ][TZPUQ]^Q\] #DnC$& [TZ][TZ^\UQ]^Q\]& XQ^TcW[TZ][TZYM^Q]& [TZ][TZ\MXUPM^Q& MYP [TZ][TZ\Z^TUZM^Q] #DnF$( Representative non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2'Cg$3 MYP B&Bo'PUXQ^TcWTcP\MdUYQ #g7<2- N(CH3)-N(CH3)-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotides. In certain embodiments, linkages having a chiral atom can be prepared as racemic mixtures, as separate enantomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known to those skilled in the art. The phosphate group in the linking group can be modified by replacing one of the oxygens with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some Docket No.: 121301-22520 ALN-511-WO embodiments, one of the non-bridging phosphate oxygen atoms in the linkage can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an aryl group, etc…), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral; in other words a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers. Thus, while not wishing to be bound by theory, modifications to both non-bridging oxygens, which eliminate the chiral center, e.g. phosphorodithioate formation, can be desirable in that they cannot produce diastereomer mixtures. Thus, the non-bridging oxygens can be independently any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl). The phosphate linker can also be modified by replacement of bridging oxygen, (i.e. oxygen that links the phosphate to the sugar of the monomer), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at the either one of the linking oxygens or at both linking oxygens. When the bridging oxygen is the 3’-oxygen of a nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5’-oxygen of a nucleoside, replacement with nitrogen is preferred. Modified phosphate linkages where at least one of the oxygen linked to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorous group, are also referred to as “non-phosphodiester intersugar linkage” or “non-phosphodiester linker.” In certain embodiments, the phosphate group can be replaced by non-phosphorus containing connectors, e.g. dephospho linkers. Dephospho linkers are also referred to as non-phosphodiester linkers herein. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety. Examples of moieties which can replace the phosphate group include, but are not limited to, amides (for example amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker, sulfide,sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’, C3’-CH2-NH-NH-C5’, 3'- NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5’ and nonionic linkages containing mixed N, O, S Docket No.: 121301-22520 ALN-511-WO and CH2 component parts. See for example, Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp.40-65). Preferred embodiments include methylenemethylimino (MMI),methylenecarbonylamino, amides,carbamate and ethylene oxide linker. One skilled in the art is well aware that in certain instances replacement of a non-bridging oxygen can lead to enhanced cleavage of the intersugar linkage by the neighboring 2’-OH, thus in many instances, a modification of a non-bridging oxygen can necessitate modification of 2’-OH, e.g., a modification that does not participate in cleavage of the neighboring intersugar linkage, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA. Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Sp isomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Rp isomer, phosphorodithioates, phsophotriesters, aminoalkylphosphotrioesters, alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidate), and boranophosphonates. In some embodiments, the dsRNA agent of the disclosure comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all) modified or nonphosphodiester linkages. In some embodiments, the dsRNA agent of the disclosure comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all) phosphorothioate linkages. The dsRNA agent of the disclosures can also be constructed wherein the phosphate linker and the sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone. Examples include the morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA) and backnone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate. The dsRNA agent of the disclosures described herein can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the dsRNA agent of the disclosures provided herein are all such possible isomers, as well as their racemic and optically pure forms. D. Terminal Modifications In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5’-end of the antisense strand. In one embodiment, the phosphate mimic is a 5’-vinyl phosphonate (VP). Docket No.: 121301-22520 ALN-511-WO In some embodiments, the 5’-end of the antisense strand of the dsRNA agent does not contain a 5’-vinyl phosphonate (VP). Ends of the iRNA agent of the disclosure can be modified. Such modifications can be at one end or both ends. For example, the 3^ and/or 5^ ends of an iRNA can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a linker. The terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C- 3^ or C-5^ O, N, S or C group of the sugar. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). When a linker/phosphate-functional molecular entity-linker/phosphate array is interposed between two strands of a double stranded oligomeric compound, this array can substitute for a hairpin loop in a hairpin-type oligomeric compound. Terminal modifications useful for modulating activity include modification of the 5’ end of iRNAs with phosphate or phosphate analogs. In certain embodiments, the 5’end of an iRNA is phosphorylated or includes a phosphoryl analog. Exemplary 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Modifications at the 5’-terminal end can also be useful in stimulating or inhibiting the immune system of a subject. In some embodiments, the 5’-end of the oligomeric compound comprises the modification
Figure imgf000057_0001
, wherein W, X and Y are each independently selected from the group consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR3 (R is hydrogen, alkyl, aryl), BH3-, C (i.e. an alkyl group, an aryl group, etc…), H, NR2 (R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl or aryl); A and Z are each independently for each occurrence absent, O, S, CH2, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, wherein backbone of the alkylene can comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and/or at the end; and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A is replacing the oxygen linked to 5’ carbon of sugar. When n is 0, W and Y together with the P to which they are attached can form an optionally substituted 5-8 membered heterocyclic, wherein W an Y are each independently O, S, NR’ or alkylene. Preferably the heterocyclic is substituted with an aryl or heteroaryl. In some embodiments, one or both hydrogen on C5’ of the 5’- terminal nucleotides are replaced with a halogen, e.g., F. Exemplary 5’-modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)P-O- 5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O- P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); 5'-alpha- thiotriphosphate; 5’-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'). Other 5’-modification include 5'-alkylphosphonates Docket No.: 121301-22520 ALN-511-WO (R(OH)(O)P-O-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc…), 5'-alkyletherphosphonates (R(OH)(O)P-O-5', R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc…). Other exemplary 5’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5', ((HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5', ((HO)2(X)P-[- (CH2)a-O-P(X)(OH)-O]b- 5'; dialkyl terminal phosphates and phosphate mimics: HO[-(CH2)a-O- P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a- O-P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[-(CH2)a- P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', wherein a and b are each independently 1-10. Other embodiments, include replacement of oxygen and/or sulfur with BH3, BH3- and/or Se. Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorescein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include targeting ligands. Terminal modifications can also be useful for cross-linking an oligonucleotide to another moiety; modifications useful for this include mitomycin C, psoralen, and derivatives thereof. E. Thermally Destabilizing Modifications The compounds of the disclosure, such as iRNAs or dsRNA agents, can be optimized for RNA interference by increasing the propensity of the iRNA duplex to disassociate or melt (decreasing the free energy of duplex association) by introducing a thermally destabilizing modification in the sense strand at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5’- end of the antisense strand, or at positions 2-9 of the 5’-end of the antisense strand). This modification can increase the propensity of the duplex to disassociate or melt in the seed region of the antisense strand. The thermally destabilizing modifications can include abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycerol nuceltic acid (GNA). abasic modifications are:
Figure imgf000058_0001
Docket No.: 121301-22520 ALN-511-WO The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent and/or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are or in combination absent from the nucleotide. In some embodiments, nucleotide
Figure imgf000059_0001
wherein B is a modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers with bonds between C1'-C4' being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). 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:
Figure imgf000059_0002
The thermally destabilizing modification 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. Exemplary mismatch basepairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present 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 Docket No.: 121301-22520 ALN-511-WO ribose sugars of the nucleotides. In certain embodiments, the compounds of the disclosure, such as siRNA or iRNA agent, contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand. More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety. The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications. Nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:
Figure imgf000060_0001
. Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes to natural are:
Figure imgf000060_0002
. In some embodiments, compounds of the disclosure can comprise 2’-5’ linkages (with 2’-H, 2’-OH and 2’-OMe and with P=O or P=S). For example, the 2’-5’ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5’ end of the sense strand to avoid sense strand activation by RISC. In another embodiment, compounds of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2’-H, 2’-OH and 2’-OMe). For example, these L sugar modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5’ end of the sense strand to avoid sense strand activation by RISC. In one embodimennt the iRNA agent of the disclosure is conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, Docket No.: 121301-22520 ALN-511-WO [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone. In some embodoments, at least one strand of the iRNA agent of the disclosure disclosed herein is 5’ phosphorylated or includes a phosphoryl analog at the 5’ prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O- P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7- methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O- (HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'- monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S- 5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g.5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)2(O)P- 5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-). IV. Modified RNAi agents of the Disclosure Comprising Motifs In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in U.S. Patent Nos.9,796,974 and 10,668,170, and U.S. Patent Publication Nos.2014/288158, 2018/008724, 2019/038768, and 2020/353097, the entire contents of each of which are incorporated herein by reference. As shown therein and in PCT Publication No. WO 2013/074974 (the entire contents of which are incorporated by reference), one or more motifs of three identical modifications on three consecutive nucleotides may be introduced into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand. In one embodiment, the iRNA agent of the disclosure is a double ended bluntmer of 19 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 7,8,9 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end. In one embodiment, the iRNA agent of the disclosure is a double ended bluntmer of 20 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three Docket No.: 121301-22520 ALN-511-WO consecutive nucleotides at positions 8,9,10 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end. In one embodiment, the iRNA agent of the disclosure is a double ended bluntmer of 21 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end. In one embodiment, the iRNA agent of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end; the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end, wherein one end of the iRNA is blunt, while the other end is comprises a 2 nt overhang. Preferably, the 2 nt overhang is at the 3’-end of the antisense. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3). In one embodiment, the iRNA agent of the disclosure comprises a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1- 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with sense strand, thereby forming a 3' single stranded overhang of 1-6 nucleotides; wherein the 5' terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5' overhang; wherein at least the sense strand 5' terminal and 3' terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at or near the cleavage site. In one embodiment, the iRNA agent of the disclosure comprises a sense and antisense strands, wherein said iRNA agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at position 11,12,13 from the 5’ end; wherein said 3’ end of said first strand and said 5’ end of said second strand form a blunt end and said second strand is 1-4 nucleotides longer at its 3’ end than the first strand, wherein the Docket No.: 121301-22520 ALN-511-WO duplex region region which is at least 25 nucleotides in length, and said second strand is sufficiently complemenatary to a target mRNA along at least 19 nt of said second strand length to reduce target gene expression when said iRNA agent is introduced into a mammalian cell, and wherein dicer cleavage of said iRNA preferentially results in an siRNA comprising said 3’ end of said second strand, thereby reducing expression of the target gene in the mammal. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3). In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand. For instance, the sense strand can contain at least one motif of three 2’-F modifications on three consecutive nucleotides within 7-15 positions from the 5’end. In one embodiment, the antisense strand of the iRNA agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand. For instance, the antisense strand can contain at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides within 9-15 positions from the 5’end. For iRNA agent having a duplex region of 17-23 nt in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5’-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1st nucleotide from the 5’-end of the antisense strand, or, the count starting from the 1st paired nucleotide within the duplex region from the 5’- end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the iRNA from the 5’-end. In some embodiments, the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand also contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand. The modification in the motif occurring at or near the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand. In some embodiments, the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand. In one embodiment, the antisense strand also contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at or near the cleavage site. In some embodiments, the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end, and wherein the Docket No.: 121301-22520 ALN-511-WO antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end. In one embodiment, the iRNA agent of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings. In one embodiment, the iRNA agent of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5’- end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex. In one embodiment, the nucleotide at the 1 position within the duplex region from the 5’-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. In another embodiment, the nucleotide at the 3’-end of the sense strand is deoxythimidine (dT). In another embodiment, the nucleotide at the 3’-end of the antisense strand is deoxythimidine (dT). In one embodiment, there is a short sequence of deoxythimidine nucleotides, for example, two dT nucleotides on the 3’-end of the sense or antisense strand. In certain embodiments, the compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a 5’-vinyl phosphonate modified nucleotide of the disclosure has the structure:
Figure imgf000064_0001
wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C1-20alkoxy (e.g., methoxy or n-hexadecyloxy); R5’ is =C(H)-P(O)(OH)2 and the double bond between the C5’ carbon and R5’ is in the E or Z orientation (e.g., E orientation); and Docket No.: 121301-22520 ALN-511-WO B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil. A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5’ end of the antisense strand of the dsRNA. Vinyl phosphonate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure includes the preceding structure, where R5’ is =C(H)-OP(O)(OH)2 and the double bond between the C5’ carbon and R5’ is in the E or Z orientation (e.g., E orientation). In one aspect, the disclosure relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene having reduced off-target effects as described in U.S. Patent Nos. 10,233448, 10,612,024, and 10,612,027, and U.S. Patent Publication Nos.2017/275626, 2019/241891, 2019/241893, and 2021/017519, the entire contents of each of which are incorporated herein by reference. As exemplified therein, a motif comprising, e.g., a thermally destabilizing nucleotide, e.g., i) a nucleotide that forms a mismatch pair with the opposing nucleotide in the antisense strand, ii) a nucleotide having an abasic modification, and/or iii) a nucleotide having a sugar modification, and placed at a site opposite to the seed region (positions 2-8) may be introduced into the sense strand. In one embodiment, the dsRNA agent of the disclosure does not contain any 2’-F modification. In one embodiment, the dsRNA agent of the disclosure only contains 2’-F and 2’-OMe modifications. In one embodiment, the sense strand and/or antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages. In one embodiment, each of the sense and antisense strands of the dsRNA agent has 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides, and the antisense strand has 19- 25 nucleotides. In another example, the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides. In one embodiment, the nucleotide at position 1 of the 5’-end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pair from the 5’-end of the antisense strand is an AU base pair. In one embodiment, the antisense strand of the dsRNA agent of the disclosure is 100% complementary to a target RNA to hybridize thereto and inhibits its expression through RNA interference. In another embodiment, the antisense strand of the dsRNA agent of the disclosure is at Docket No.: 121301-22520 ALN-511-WO least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to a target RNA. In one aspect, the disclosure relates to a dsRNA agent as defined herein capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermally destabilizing nucleotide, wherein at least one of said thermally destabilizing nucleotide occurs at or near the site that is opposite to the seed region of the antisense strand (i.e. at position 2-8 of the 5’-end of the antisense strand, or at positions 2-9 of the 5’-end of the antisense strand). Each of the embodiments and aspects described in this specification relating to the dsRNA represented by formula (I) can also apply to the dsRNA containing the thermally destabilizing nucleotide. The thermally destabilizing nucleotide can occur, for example, between positions 14-17 of the 5’-end of the sense strand when the sense strand is 21 nucleotides in length. The antisense strand contains at least two modified nucleic acids that are smaller than a sterically demanding 2’-OMe modification. Preferably, the two modified nucleic acids that are smaller than a sterically demanding 2’-OMe are separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 of the 5’end of the antisense strand. In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand is one or more GalNAc derivatives attached a bivalent or trivalent branched linker, such as:
Figure imgf000066_0001
one example, the ASGPR ligand is attached to the 3’ end of the sense strand. For example, the dsRNA agent as defined herein can comprise i) a phosphorus-containing group at the 5’-end of the sense strand or antisense strand; ii) with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’- end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand); and iii) a ligand, such as a ASGPR ligand (e.g., one or more GalNAc derivatives) at 5’-end or 3’-end of the sense strand or antisense strand. For instance, the ligand may be at the 3’-end of the sense strand. In a particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; and (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2’- OMe 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’-OMe modifications at positions 1, 3, Docket No.: 121301-22520 ALN-511-WO 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2’F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5’ end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2’- OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5’ end); and (iv) 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’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2’-F modifications at positions 7, and 9, and a desoxy-nucleotide (e.g. dT) at position 11 (counting from the 5’ end); and (iv) 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’-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2’-F 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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2’-F modifications at positions 7, 9, 11, 13, and 15; and (iv) 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’-OMe modifications at Docket No.: 121301-22520 ALN-511-WO positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 9, and 12 to 21, and 2’-F modifications at positions 10, and 11; and (iv) 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’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2’-OMe modifications at positions 2, 4, 6, 8, 12, and 14 to 21; and (iv) 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’-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 5, Docket No.: 121301-22520 ALN-511-WO 7, 8, 10 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 9, 14, and 16 (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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 8, 9, 14, and 16 (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 agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another particular embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 19 nucleotides; (ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2’-F modifications at positions 5, and 7 to 9; and (iv) 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 21 nucleotides; (ii) 2’-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2’-F modifications at positions 2, 6, 8, 9, 14, and 16 (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 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In one embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 18-23 nucleotides; (ii) three consecutive 2’-F modifications at positions 7-15; and (b) an antisense strand having: (i) a length of 18-23 nucleotides; (ii) at least 2’-F modifications anywhere on the strand; and (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides Docket No.: 121301-22520 ALN-511-WO overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex. In one embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 18-23 nucleotides; (ii) less than four 2’-F modifications; (b) an antisense strand having: (i) a length of 18-23 nucleotides; (ii) less than twelve 2’-F modfication; and (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex. In one embodiment, the dsRNA agents of the present disclosure comprise: (a) a sense strand having: (i) a length of 19-35 nucleotides; (ii) less than four 2’-F modifications; (b) an antisense strand having: (i) a length of 19-35 nucleotides; (ii) less than twelve 2’-F modfication; and (iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); and wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex. In another embodiment, the dsRNA agents of the present disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) a liophilic moiety (e.g., a C22 hydrocarbon chain); (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 4, 6, 8 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 14, and 16, a 2’-deoxy-modified nucleotide at position 5, and a GNA at position 7 (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 RNAi agents have a two-nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In another embodiment, the dsRNA agents of the present disclosure comprises: (a) a sense strand having: (i) a length of 21 nucleotides; (ii) a liophilic moiety (e.g., a C22 hydrocarbon chain); (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and (iv) 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’-OMe modifications at positions 1, 3 to 5, 7 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 14, and 16 (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 Docket No.: 121301-22520 ALN-511-WO 23 (counting from the 5’ end); wherein the RNAi agents have a two-nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand. In one embodiment, the dsRNA agents of the present disclosure comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have less than 20% , less than 15% and less than 10% non-natural nucleotide. Examples of non-natural nucleotide includes acyclic nucleotides, LNA, HNA, CeNA, 2’- methoxyethyl, 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), a 2'- O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F, and others. In one embodiment, the dsRNA agents of the present disclosure comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have greater than 80% , greater than 85% and greater than 90% natural nucleotide, such as 2’-OH, 2’-deoxy and 2’-OMe are natural nucleotides. In one embodiment, the dsRNA agents of the present disclosure comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have 100% natural nucleotide, such as 2’-OH, 2’-deoxy and 2’-OMe are natural nucleotides. Various publications described multimeric siRNA and can all be used with the iRNA of the disclosure. Such publications include WO2007/091269, US Patent No.7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520, which are hereby incorporated by reference in their entirety. In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the iRNA agent of the disclosure is modified. In some embodiments, each of the sense and antisense strands of the iRNA agent is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2’-methoxyethyl, 2’- O-methyl, 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), a 2'-O- dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F. In some embodiments, each of the sense and antisense strands of the iRNA agent contains at least two different modifications. Docket No.: 121301-22520 ALN-511-WO In some embodiments, the dsRNA agent of the disclosure contains one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 2’-F modification(s). In one example, dsRNA agent of the disclosure contains nine or ten 2’-F modifications. The iRNA agent of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, the iRNA comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paried nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3’-end of the antisense strand. In some embodiments, the sense strand and/or antisense strand of the iRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages. . In one aspect, the disclosure relates to a iRNA agent capable of inhibiting the expression of a target gene. The iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermally destabilizing nucleotide, wherein at at least one said thermally destabilizing nucleotide occurs at or near the site that is opposite to the seed region of the antisense strand (i.e .at position 2-8 of the 5’-end of the antisense strand, or at Docket No.: 121301-22520 ALN-511-WO positions 2-9 of the 5’-end of the antisense strand). For example, the thermally destabilizing nucleotide occurs between positions 14-17 of the 5’-end of the sense strand when the sense strand is 21 nucleotides in length. The antisense strand contains at least two modified nucleic acids that are smaller than a sterically demanding 2’-OMe modification. Preferably, the two modified nucleic acids that is smaller than a sterically demanding 2’-OMe are separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 of the 5’end of the antisense strand. In some embodiments, the compound of the disclosure disclosed herein is a miRNA mimic. In one design, miRNA mimics are double stranded molecules (e.g., with a duplex region of between about 16 and about 31 nucleotides in length) and contain one or more sequences that have identity with the mature strand of a given miRNA. Double-stranded miRNA mimics have designs similar to as described above for double-stranded iRNAs. In some embodiments, a miRNA mimic comprises a duplex region of between 16 and 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide), and all of the Cs and Us; the antisense strand modifications can comprise 2' F modification of all of the Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with a 2 nucleotide 3 ' overhang. V. Lipohilic Moieties A lipohilic moiety of the disclosure can comprise at least one C10-C26 hydrocarbon chain. As described In U.S. Provisional Application No.63/255,984, filed on October 15, 2021 (the entire contents of which are incorporated herein by reference), including a C22 hydrocarbon chain, e.g., saturated or unsaturated, on one or more internal position(s) of the dsRNA agent increases lipophilicity of the dsRNA agent and provides optimal hydrophobicity for the enhanced in vivo delivery of dsRNA to, e.g., muscle tissue and/or adipose 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 laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci.41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its logKow exceeds 0. Typically, the lipophilic moiety possesses a logKow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the logKow of 6- amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the logKow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7. Docket No.: 121301-22520 ALN-511-WO 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 C22 hydrocarbon chain can increase or decrease the partition coefficient (e.g., logKow) value of the C22 hydrocarbon chain. Alternatively, the hydrophobicity of the dsRNA agent, conjugated to one or more C22 hydrocarbon chains, can be measured by its protein binding characteristics. For instance, 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. In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the dsRNA agent, measured by fraction of unbound dsRNA 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 siRNA. In certain embodiments, the one or more C22 hydrocarbon chains is an aliphatic, alicyclic, or polyalicyclic compound is an aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compound. The hydrocarbon chain may comprise various substituents and/or one or more heteroatoms, such as an oxygen or nitrogen atom. The one or more C22 hydrocarbon chains may be attached to the iRNA agent by any method known in the art, including via a functional grouping already present in the lipophilic moiety or introduced into the iRNA agent, such as a hydroxy group (e.g., —CO—CH2—OH). The functional groups already present in the C22 hydrocarbon chain 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 C22 hydrocarbon chain 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 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. In some embodiments, the C22 hydrocarbon chain is conjugated to the dsRNA agent via a linker 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. In one embodiment, the one or more C22 hydrocarbon chains is a C22 acid, e.g., the C22 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. Docket No.: 121301-22520 ALN-511-WO
Figure imgf000075_0001
In one embodiment, the one or more C22 hydrocarbon chains is a C22 alcohol, e.g. the C22 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, and cis-4,7,10,13,16,19-docosahexanol.
Figure imgf000075_0002
In one embodiment, the one or more C22 hydrocarbon chains is not cis-4,7,10,13,16,19- docosahexanoic acid. In one embodiment, the one or more C22 hydrocarbon chains is not cis- 4,7,10,13,16,19-docosahexanol. In one embodiment, the one or more C22 hydrocarbon chains is not cis-4,7,10,13,16,19-docosahexanoic acid and is not cis-4,7,10,13,16,19-docosahexanol. In one embodiment, the one or more C22 hydrocarbon chains is a C22 amide, e.g., the C22 amide is selected 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. In one embodiment, the C22 hydrocarbon chain includes, but are not limited to, docosan-2-yl, 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- (butyl)octadecan-1-yl, 2-(propyl)nonadecan-1-yl, 2-(ethyl)eicosan-1-yl, 2-(methyl)henicosan-1-yl, 3- (nonyl)tridecan-1-yl, 3-(octyl)tetradecan-1-yl, 3-(heptyl)pentadecan-1-yl, 3-(hexyl)hexadecan-1-yl, 3- (pentyl)heptadecan-1-yl, 3-(butyl)octadecan-1-yl, 3-(propyl)nonadecan-1-yl, 3-(ethyl)eicosan-1-yl, 3- Docket No.: 121301-22520 ALN-511-WO (methyl)henicosan-1-yl, 4-(octyl)tetradecan-1-yl, 4-(heptyl)pentadecan-1-yl, 4-(hexyl)hexadecan-1-yl, 4-(pentyl)heptadecan-1-yl, 4-(butyl)octadecan-1-yl, 4-(propyl)nonadecan-1-yl, 4-(ethyl)eicosan-1-yl, 4-(methyl)henicosan-1-yl, 5-(heptyl)pentadecan-1-yl, 5-(hexyl)hexadecan-1-yl, 5-(pentyl)heptadecan- 1-yl, 5-(butyl)octadecan-1-yl, 5-(propyl)nonadecan-1-yl, 5-(ethyl)eicosan-1-yl, 5-(methyl)henicosan- 1-yl, 6-(hexyl)hexadecan-1-yl, 6-(pentyl)heptadecan-1-yl, 6-(butyl)octadecan-1-yl, 6- (propyl)nonadecan-1-yl, 6-(ethyl)eicosan-1-yl, 6-(methyl)henicosan-1-yl, 7-(pentyl)heptadecan-1-yl, 7-(butyl)octadecan-1-yl, 7-(propyl)nonadecan-1-yl, 7-(ethyl)eicosan-1-yl, 7-(methyl)henicosan-1-yl, 8-(butyl)octadecan-1-yl, 8-(propyl)nonadecan-1-yl, 8-(ethyl)eicosan-1-yl, 8-(methyl)henicosan-1-yl, 9-(propyl)nonadecan-1-yl, 9-(ethyl)eicosan-1-yl, 9-(methyl)henicosan-1-yl, 10-(ethyl)eicosan-1-yl, 10-(methyl)henicosan-1-yl, and 11-(methyl)henicosan-1-yl, which is substituted at the 2’-oxygen of a nucleoside of an oligonucleotide herein. In certain embodiments, more than one C22 hydrocarbon chains can be incorporated into the double-strand iRNA agent, particularly when the C22 hydrocarbon chains has a low lipophilicity or hydrophobicity. In one embodiment, two or more C22 hydrocarbon chains are incorporated into the same strand of the double-strand iRNA agent. In one embodiment, each strand of the double-strand iRNA agent has one or more C22 hydrocarbon chains incorporated. In one embodiment, two or more C22 hydrocarbon chains are incorporated into the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the double-stranded iRNA agent. This can be achieved by, e.g., conjugating the two or more aturated or unsaturated C22 hydrocarbon chains via a carrier, and/or conjugating the two or more C22 hydrocarbon chains via a branched linker, and/or conjugating the two or more C22 hydrocarbon chains via one or more linkers, with one or more linkers linking the C22 hydrocarbon chains consecutively. The one or more C22 hydrocarbon chains may be conjugated to the iRNA agent via a direct attachment to the ribosugar of the iRNA agent. Alternatively, the one or more C22 hydrocarbon chains may be conjugated to the double-strand iRNA agent via a linker or a carrier. In certain embodiments, the one or more C22 hydrocarbon chains may be conjugated to the iRNA agent via one or more linkers (tethers). In one embodiment, the one or more C22 hydrocarbon chains is conjugated to the 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. In one embodiment, the lipophilic moiety (e.g., C22 hydrocarbon chain) is attached at the 2’ position on an internal nucleotide of the sense or antisense strand. In one embodiment, the lipophilic moiety (e.g., C22 hydrocarbon chain) is attached at the 2’ position on the nucleotide at any of positions 4-8 and 13-18 on the sense strand (e.g., position 4, 5, 6, 7, 8, 13, 14, 15, 16, 17, 18) counting from the 5’ end. In one embodiment, the lipophilic moiety (e.g., C22 hydrocarbon chain) is attached at the 2’ position on the nucleotide at position 6 or 16 on the sense strand counting from the 5’ end. A. Linkers/Tethers Docket No.: 121301-22520 ALN-511-WO Linkers/Tethers are connected to the one or more C22 hydrocarbon chains at a “tethering attachment point (TAP).” Linkers/Tethers may include any C1-C100 carbon-containing 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 least one nitrogen atom. In certain embodiments, 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 (underlined) 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- C(O)-O-; TAP-C(O)-(CH2)n-NH-C(O)-; TAP-C(O)-(CH2)n-; TAP-C(O)-NH-; TAP-C(O)-; TAP- (CH2)n-C(O)-; TAP-(CH2)n-C(O)O-; TAP-(CH2)n-; or 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. Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or hydrazino group, -NHNH2. The linker/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. 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- 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)(LIGAND); TAP-C(O)- (CH2)n(LIGAND); TAP-C(O)-NH(LIGAND); TAP-C(O)(LIGAND); TAP-(CH2)n-C(O) (LIGAND); TAP-(CH2)n-C(O)O(LIGAND); TAP-(CH2)n(LIGAND); or TAP-(CH2)n-NH-C(O)(LIGAND). In some embodiments, amino terminated linkers/tethers (e.g., NH2, ONH2, NH2NH2) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, amino terminated linkers/tethers (e.g., NH2, ONH2, NH2NH2) can acylated, e.g., with C(O)CF3. In some embodiments, the linker/ tether can terminate with a mercapto group (i.e., SH) or an olefin (e.g., CH=CH2). For example, the tether can be TAP-(CH2)n-SH, TAP-C(O)(CH2)nSH, TAP- (CH2)n-(CH=CH2), or TAP-C(O)(CH2)n(CH=CH2), in which n can be as described elsewhere. The 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. In other embodiments, 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, 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 (underlined) 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 TAP-NR’’’’(CH2) nC(O) OC6F5, in which n is 1-11 and R’’’’ is C1-C6 alkyl; or -(CH2)nCH2LG; TAP- C(O)(CH2)nCH2LG; or TAP-NR’’’’(CH2)nCH2LG, in which n can be as described elsewhere and Docket No.: 121301-22520 ALN-511-WO R’’’’ is C1-C6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). 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. In other it can be desirable for the monomer to include a phthalimido group (K) at the terminal position of the
Figure imgf000078_0001
In other embodiments, other protected amino groups can be at the terminal position of the 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). Any of the linkers/tethers described herein may further include one or more additional linking groups, e.g., -O-(CH2)n-, -(CH2)n-SS-, -(CH2)n-, or -(CH=CH)-. B. Cleavable linkers/tethers In some embodiments, 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. In one embodiment, at least one of the linkers/tethers can be a reductively cleavable linker (e.g., a disulfide group). In one embodiment, 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., 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. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases. A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some tethers will have a linkage group that is cleaved at a preferred pH, thereby Docket No.: 121301-22520 ALN-511-WO 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 chemical junction (e.g., a linking group) that links a ligand to an iRNA agent can include 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 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. For example, an iRNA agent that targets an mRNA in liver cells can be conjugated to a tether that includes an ester group. Liver cells are rich in esterases, and therefore the tether will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Cleavage of the tether releases the iRNA agent from a ligand that is attached to the distal end of the tether, thereby potentially enhancing silencing activity of the iRNA agent. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis. Tethers that contain peptide bonds can be conjugated to iRNA agents target to cell types rich in peptidases, such as liver cells and synoviocytes. For example, an iRNA agent targeted to synoviocytes, such as for the treatment of an inflammatory disease (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond. In general, 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. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. 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. In preferred embodiments, 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). C. Redox Cleavable Linking Groups 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—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA Docket No.: 121301-22520 ALN-511-WO moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media. D. Phosphate-Based Cleavable Linking Groups Phosphate-based linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of 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(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, — S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S— P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O— P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above. E. Acid Cleavable Linking Groups Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, ketals, acetals, esters, and esters of amino acids. Acid OWQM`MNWQ S\Z_[] OMY TM`Q ^TQ SQYQ\MW RZ\X_WM g7nBBg& 7#C$C& Z\ gC7#C$( 5 [\QRQ\\QP 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. F. Ester-Based Linking Groups Docket No.: 121301-22520 ALN-511-WO Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula — C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above. G. Peptide-Based Cleaving Groups 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 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 —NHCHR1C(O)NHCHR2C(O)—, where R1 and R2 are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. H. Biocleavable linkers/tethers The linkers can also includes biocleavable linkers that are nucleotide and non-nucleotide linkers or combinations thereof that connect two parts of a molecule, for example, one or both strands of two individual siRNA molecules to generate a bis(siRNA). In some embodiments, mere electrostatic or stacking interaction between two individual siRNAs can represent a linker. The non- nucleotide linkers include tethers or linkers derived from monosaccharides, disaccharides, oligosaccharides, and derivatives thereof, aliphatic, alicyclic, hetercyclic, and combinations thereof. In some embodiments, at least one of the linkers (tethers) 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. In one embodiment, 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: Docket No.: 121301-22520 ALN-511-WO
Figure imgf000082_0001
Docket No.: 121301-22520 ALN-511-WO
Figure imgf000083_0001
Docket No.: 121301-22520 ALN-511-WO
Figure imgf000084_0001
More discussion about the biocleavable linkers may be found in PCT application No. PCT/US18/14213, entitled “Endosomal Cleavable Linkers,” filed on January 18, 2018, the entire contents of which are incorporated herein by reference. I. Carriers In certain embodiments, the one or more C22 hydrocarbon chains is conjugated to the iRNA agent via a carrier that replaces one or more nucleotide(s). The carrier can be a cyclic group or an acyclic group. In one embodiment, 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. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone. In some embodiments, 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 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 Docket No.: 121301-22520 ALN-511-WO pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is 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 attached to the carrier an as described above.
Figure imgf000085_0001
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. Alternatively, 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 present in an iRNA agent. a. Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated Monomers (Cyclic) Cyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) provided below (in that structure preferred backbone attachment points can be chosen from R1 or R2; R3 or R4; or R9 and R10 if Y is CR9R10 (two positions are chosen to give two backbone attachment points, e.g., R1 and R4, or R4 and R9)). Preferred tethering attachment points include R7; R5 or R6 when X is CH2. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R1 or R2; R3 or R4; or R9 or R10 (when Y is CR9R10), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be -CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom. Docket No.: 121301-22520 ALN-511-WO
Figure imgf000086_0001
(LCM-2) wherein: X is N(CO)R7, NR7 or CH2; Y is NR8, O, S, CR9R10; Z is CR11R12 or absent; Each of R1, R2, R3, R4, R9, and R10 is, independently, H, ORa, or (CH2)nORb, provided that at least two of R1, R2, R3, R4, R9, and R10 are ORa and/or (CH2)nORb; Each of R5, R6, R11, and R12 is, independently, a ligand, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14; R7 can be a ligand, e.g., R7 can be Rd , or R7 can be a ligand tethered indirectly to the carrier, e.g., through a tethering moiety, e.g., C1-C20 alkyl substituted with NRcRd; or C1-C20 alkyl substituted with NHC(O)Rd; R8 is H or C1-C6 alkyl; R13 is hydroxy, C1-C4 alkoxy, or halo; R14 is NRcR7; R15 is C1-C6 alkyl optionally substituted with cyano, or C2-C6 alkenyl; R16 is C1-C10 alkyl; R17 is a liquid or solid phase support reagent; L is -C(O)(CH2)qC(O)-, or -C(O)(CH2)qS-; Ra is a protecting group, e.g., CAr3; (e.g., a dimethoxytrityl group) or Si(X5’)(X5”)(X5”’) in which (X5’),(X5”), and (X5”’) are as described elsewhere. Rb is P(O)(O-)H, P(OR15)N(R16)2 or L-R17; Rc is H or C1-C6 alkyl; Rd is H or a ligand; Each Ar is, independently, C6-C10 aryl optionally substituted with C1-C4 alkoxy; n is 1-4; and q is 0-4. Exemplary carriers include those in which, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent; or X is N(CO)R7 or NR7, Y is CR9R10, and Z is CR11R12; or X is N(CO)R7 or NR7, Y is NR8, and Z is CR11R12; or X is N(CO)R7 or NR7, Y is O, and Z is CR11R12; or X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C5 cycloalkyl (H, z = 1). In certain embodiments, the carrier may be based on the pyrroline ring system or the 4- hydroxyproline ring system, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent (D). Docket No.: 121301-22520 ALN-511-WO
Figure imgf000087_0001
. OFG1 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 (- CH2OFG1 in D). OFG2 is preferably attached directly to one of the carbons in the five-membered ring (-OFG2 in D). For the pyrroline-based carriers, -CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-3; or -CH2OFG1 may be attached to C-3 and OFG2 may be attached to C-4. In certain embodiments, CH2OFG1 and OFG2 may be geminally substituted to one of the above-referenced carbons. For the 3-hydroxyproline-based carriers, -CH2OFG1 may be attached to C-2 and OFG2 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, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1 and OFG2 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. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 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:
Figure imgf000087_0002
. Docket No.: 121301-22520 ALN-511-WO In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is
Figure imgf000088_0001
N(CO)R7 or NR7, Y is CR9R10, and Z is CR11R12. . OFG1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [-(CH2)nOFG1 in E]. OFG2 is preferably attached directly to one of the carbons in the six- membered ring (-OFG2 in E). -(CH2)nOFG1 and OFG2 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. Alternatively, - (CH2)nOFG1 and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH2)nOFG1 may be attached to C-2 and OFG2 may be attached to C-3; -(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-2; - (CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or -(CH2)nOFG1 may be attached to C-4 and OFG2 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 linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)nOFG1 and OFG2 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. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 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. In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is (G), e.g., X is N(CO)R7
Figure imgf000088_0002
OFG1 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 (-CH2OFG1 in F or G). OFG2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG2 in F or G). For both F and G, -CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG1 and OFG2 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 Docket No.: 121301-22520 ALN-511-WO bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1 and OFG2 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. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 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). R’’’ can be, e.g., C1- C6 alkyl, preferably CH3. The tethering attachment point is preferably nitrogen in both F and G. In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH2; Y
Figure imgf000089_0001
methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [- (CH2)nOFG1 in H]. OFG2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG2 in H). -(CH2)nOFG1 and OFG2 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. Alternatively, -(CH2)nOFG1 and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH2)nOFG1 may be attached to C-2 and OFG2 may be attached to C-3; - (CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-2; -(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or -(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-3; -(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-5; or - (CH2)nOFG1 may be attached to C-5 and OFG2 may be attached to C-4. The decalin or indane-based monomers may 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. Thus, - (CH2)nOFG1 and OFG2 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. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 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). In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7. Docket No.: 121301-22520 ALN-511-WO
Figure imgf000090_0001
Other carriers may include those based on 3-hydroxyproline (J). . Thus, -(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another. 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. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 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 U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties. b. Sugar Replacement-Based Monomers (Acyclic) Acyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as ribose replacement monomer subunit (RRMS) monomer Preferred carriers can have formula LCM-3 or LCM-4:
Figure imgf000090_0002
. In some embodiments, each of x, y, and z can be, independently of one another, 0, 1, 2, or 3. In formula LCM-3, when y and z are different, then the tertiary carbon can have either the R or S configuration. In preferred embodiments, 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. Each of 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 entireties. The one or more C22 hydrocarbon chains is conjugated to one or more internal positions on at least one 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 position 1 counting from the 5’ end). In one embodiment, the one or more C22 hydrocarbon chains is 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 Docket No.: 121301-22520 ALN-511-WO and positions 1 and 2 counting from the 5’ end). In one embodiment, the one or more C22 hydrocarbon chains 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). In one embodiment, the one or more C22 hydrocarbon chains is conjugated to one or more internal positions on at least one strand, except the cleavage site region of the sense strand, for instance, the one or more C22 hydrocarbon chains is not conjugated to positions 9-12 counting from the 5’-end of the sense strand, for example, the one or more C22 hydrocarbon chains is not conjugated to positions 9-11 counting from the 5’-end of the sense strand. Alternatively, the internal positions exclude positions 11-13 counting from the 3’-end of the sense strand. In one embodiment, the one or more C22 hydrocarbon chains is 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. In one embodiment, the one or more C22 hydrocarbon chains is 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. In one embodiment, the one or more C22 hydrocarbon chains is conjugated to 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 one or more C22 hydrocarbon chains is conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’end of each strand. In one embodiment, the one or more C22 hydrocarbon chains is conjugated to position 6 on the sense strand, counting from the 5’end of each strand. In one embodiment, the one or more C22 hydrocarbon chains is conjugated to position 16 on the sense strand, counting from the 5’end of each strand. In some embodiments, the one or more C22 hydrocarbon chains is conjugated to a nucleobase, sugar moiety, or internucleosidic phosphate linkage of the dsRNA agent. VI. Synthesis of RNAi Agents of the Disclosure The nucleic acids featured in the disclosure can be synthesized or modified by methods well 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. An siRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage. A. Organic Synthesis Docket No.: 121301-22520 ALN-511-WO An siRNA can be made by separately synthesizing a single stranded RNA molecule, or each respective strand of a double-stranded RNA molecule, after which the component strands can then be annealed. A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given siRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the siRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection. Organic synthesis can be used to produce a discrete siRNA species. The complementary of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini. B. dsiRNA Cleavage siRNAs can also be made by cleaving a larger siRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used: 1. In vitro transcription. dsiRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe™ RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsiRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsiRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsiRNA is produced. Similar methods using PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) can also be dotoxins that may contaminate preparations of the recombinant enzymes. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes. 2. In vitro Cleavage. dsRNA is cleaved in vitro into siRNAs, for example, using a Dicer or comparable RNAse III- based activity. For example, the dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g., a purified RNAse or RISC complex (RNA-induced silencing complex ). See, e.g., Ketting et al. Genes Dev 2001 Oct 15;15(20):2654-9 and Hammond Science 2001 Aug 10;293(5532):1146-50. dsiRNA cleavage generally produces a plurality of siRNA species, each being a particular 21 to 23 nt fragment of a source dsiRNA molecule. For example, siRNAs that include sequences Docket No.: 121301-22520 ALN-511-WO complementary to overlapping regions and adjacent regions of a source dsiRNA molecule may be present. Regardless of the method of synthesis, the siRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For example, the siRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried siRNA can then be resuspended in a solution appropriate for the intended formulation process. C. Making dsRNA agents conjugated to one or more C22 hydrocarbon chains In some embodiments, the one or more C22 hydrocarbon chains is conjugated to the dsRNA agent via a nucleobase, sugar moiety, or internucleosidic linkage. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a C22 hydrocarbon chain. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a C22 hydrocarbon chain. When one or more C22 hydrocarbon chains is conjugated to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. In one embodiment, the one or more C22 hydrocarbon chains may be conjugated to a nucleobase via a linker containing an alkyl, alkenyl or amide linkage. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Exemplary carbon atoms of a sugar moiety that the one or more C22 hydrocarbon chains can be attached to include the 2', 3', and 5' carbon atoms. The one or more C22 hydrocarbon chains can also be attached to the 1' position, such as in an abasic residue. In one embodiment, the the one or more C22 hydrocarbon chains may be conjugated to a sugar moiety, via a 2’-O modification, with or without a linker. Internucleosidic linkages can also bear the one or more C22 hydrocarbon chains. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like), the the one or more C22 hydrocarbon chains can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide- containing internucleosidic linkages (e.g., PNA), the the one or more C22 hydrocarbon chains can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom. There are numerous methods for preparing conjugates of oligonuclotides. Generally, an oligonucleotide is attached to a conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, and the like) on the oligonucleotide with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic and the other is nucleophilic. For example, an electrophilic group can be a carbonyl-containing functionality and a nucleophilic group can be an amine or thiol. Methods for conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in the literature such as, for Docket No.: 121301-22520 ALN-511-WO example, in Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety. In one embodiment, a first (complementary) RNA strand and a second (sense) RNA strand can be synthesized separately, wherein one of the RNA strands comprises a pendant C22 hydrocarbon chain, and the first and second RNA strands can be mixed to form a dsRNA. The step of synthesizing the RNA strand preferably involves solid-phase synthesis, wherein individual nucleotides are joined QYP ^Z QYP ^T\Z_ST ^TQ RZ\XM^UZY ZR UY^Q\Y_OWQZ^UPQ ,o'.o [TZ][TZPUQ]^Q\ NZYP] UY OZY]QO_^U`Q synthesis cycles. In one embodiment, the C22 hydrocarbon chain having a phosphoramidite group is coupled to ^TQ ,j'QYP Z\ .o'QYP ZR QU^TQ\ ^TQ RU\]^ #OZX[WQXQY^M\c$ Z\ ]QOZYP #]QY]Q$ EB5 ]^\MYP UY ^TQ WM]^ synthesis cycle. In the solid-phase synthesis of an RNA, the nucleotides are initially in the form of nucleoside phosphoramidites. In each synthesis cycle, a further nucleoside phosphoramidite is linked to the -OH group of the previously incorporated nucleotide. If the the one or more C22 hydrocarbon chains has a phosphoramidite group, it can be coupled in a manner similar to a nucleoside phosphoramidite to the free OH end of the RNA synthesized previously in the solid-phase synthesis. The synthesis can take place in an automated and standardized manner using a conventional RNA synthesizer. Synthesis of the molecule having the phosphoramidite group may include phosphitylation of a free hydroxyl to generate the phosphoramidite group. Synthesis procedures of the one or more C22 hydrocarbon chain-conjugated phosphoramidites are exemplified in Example 1. In general, the oligonucleotides can be synthesized using protocols known in the art, for example, as described in Caruthers et al., Methods in Enzymology (1992) 211:3-19; WO 99/54459; Wincott et al., Nucl. Acids Res. (1995) 23:2677-2684; Wincott et al., Methods Mol. Bio., (1997) 74:59; Brennan et al., Biotechnol. Bioeng. (1998) 61:33-45; and U.S. Pat. No.6,001,311; each of which is hereby incorporated by reference in its entirety. In general, the synthesis of oligonucleotides UY`ZW`Q] OZY`QY^UZYMW Y_OWQUO MOUP [\Z^QO^UYS MYP OZ_[WUYS S\Z_[]& ]_OT M] PUXQ^TZbc^\U^cW M^ ^TQ .o' QYP& MYP [TZ][TZ\MXUPU^Q] M^ ^TQ ,o'QYP( =Y M YZY'WUXU^UYS QbMX[WQ& ]XMWW ]OMWQ ]cY^TQ]Q] M\Q conducted on a Expedite 8909 RNA synthesizer sold by Applied Biosystems, Inc. (Weiterstadt, Germany), using ribonucleoside phosphoramidites sold by ChemGenes Corporation (Ashland, Mass.). Alternatively, syntheses can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, Calif.), or by methods such as those described in Usman et al., J. Am. Chem. Soc. (1987) 109:7845; Scaringe, et al., Nucl. Acids Res. (1990) 18:5433; Wincott, et al., Nucl. Acids Res. (1990) 23:2677-2684; and Wincott, et al., Methods Mol. Bio. (1997) 74:59, each of which is hereby incorporated by reference in its entirety. The nucleic acid molecules of the present disclosure may be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al., Science (1992) 256:9923; WO 93/23569; Shabarova et al., Nucl. Acids Res. (1991) 19:4247; Bellon et al., Nucleosides & Nucleotides (1997) 16:951; Bellon et al., Bioconjugate Chem. (1997) 8:204; or by hybridization following synthesis and/or deprotection. The nucleic acid molecules can be purified by gel Docket No.: 121301-22520 ALN-511-WO electrophoresis using conventional methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water. VII. Ligands In certain embodiments, the dsRNA agent of the disclosure is further modified by covalent attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached dsRNA agent of the disclosure including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound such as an oligomeric compound. A preferred list of conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a receptor which mediates delivery to a specific tissue, e.g., liver tissue. These targeting ligands can be conjugated in combination with the one or more C22 hydrocarbon chains to enable specific systemic delivery. In one embodiment, a targeting ligand, e.g., one or more GalNAc derivatives, is conjugated to a dsRNA agent of the disclosure in combination with the one or more C22 hydrocarbon chains. In another embodiment, a targeting ligand, e.g., one or more GalNAc derivatives, is not conjugated to a dsRNA agent of the disclosure in combination with the one or more C22 hydrocarbon chains. Exemplary targeting ligands that targets the receptor mediated delivery to an adipose tissue are peptide ligands such as Angiopep-2, lipoprotein receptor related protein (LRP) ligand, bEnd.3 cell binding ligand; transferrin receptor (TfR) ligand (which can utilize iron transport system in brain and cargo transport into the brain parenchyma); manose receptor ligand (which targets olfactory ensheathing cells, glial cells), glucose transporter protein, and LDL receptor ligand. Conjugate groups amenable to the present disclosure include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49); a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3- H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777); a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an Docket No.: 121301-22520 ALN-511-WO octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923). Generally, a wide variety of entities, e.g., ligands, can be coupled to the oligomeric compounds described herein. Ligands can include naturally occurring molecules, or recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide- co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG- 5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, aptamer, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g, steroids, bile acids, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., an alpha helical peptide, amphipathic peptide, RGD peptide, cell permeation peptide, endosomolytic/fusogenic peptide), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin OZRMO^Z\]& WU[Z[ZWc]MOOTM\UPQ& MY MO^U`M^Z\ ZR [,1 A5D VUYM]Q& MY MO^U`M^Z\ ZR B:'s6& ^MbZY& vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, gamma interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and a cell-permeation agent (e.g., a.helical cell-permeation agent). Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., 8 Z\ @ [Q[^UPQ]3 l& q& Z\ r [Q[^UPQ]3 B'XQ^TcW [Q[^UPQ]3 MdM[Q[^UPQ]3 [Q[^UPQ] TM`UYS one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides. A peptidomimetic (also referred to herein as an Docket No.: 121301-22520 ALN-511-WO oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic ligand can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. Exemplary amphipathic peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins. As used herein, the term “endosomolytic ligand” refers to molecules having endosomolytic properties. Endosomolytic ligands promote the lysis of and/or transport of the composition of the disclosure, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and brached polyamines, e.g. spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, linear or branched polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids. Exemplary endosomolytic/fusogenic peptides include, but are not limited to, GALA; EALA; INF-7; Inf HA-2; diINF-7; diINF-3; GLF; GALA-INF3; INF-5, n is norleucine; JTS-1; ppTG1; ppTG20; KALA; HA; Melittin; H5WYG; and CHK6HC. Without wishing to be bound by theory, fusogenic lipids fuse with and consequently destabilize a membrane. Fusogenic lipids usually have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3- phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k- DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also refered to as XTC herein). Synthetic polymers with endosomolytic activity amenable to the present disclosure are described in U.S. Pat. App. Pub. Nos.2009/0048410; 2009/0023890; 2008/0287630; 2008/0287628; 2008/0281044; 2008/0281041; 2008/0269450; 2007/0105804; 20070036865; and 2004/0198687, contents of which are hereby incorporated by reference in their entirety. Exemplary cell permeation peptides include, but are not limited to, penetratin; Tat fragment 48-60; signal sequence based peptide; PVEC; transportan; amphiphilic model peptide; Arg9; 6MO^Q\UMW OQWW aMWW [Q\XQM^UYS [Q[^UPQ3 @@',03 OQO\Z[UY D*3 l'PQRQY]UY3 q'PQRQY]UY3 DE',23 indolicidin; RFGF; RFGF analogue; and bactenecin. Docket No.: 121301-22520 ALN-511-WO Exemplary cationic groups include, but are not limited to, protonated amino groups, derived from e.g., O-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH2)nAMINE, (e.g., AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); and NH(CH2CH2NH)nCH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino). As used herein the term “targeting ligand” refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc2 and GalNAc3 (GalNAc and multivalent GalNAc are collectively referred to herein as GalNAc conjugates); D- mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, Glucose, multivalent Glucose, multivalent fucose, glycosylated polyaminoacids and lectins. The term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule. A number of folate and folate analogs amenable to the present disclosure as ligands are described in U.S. Pat. Nos.2,816,110; 5,552,545; 6,335,434 and 7,128,893, contents of which are herein incorporated in their entireties by reference. As used herein, the terms “PK modulating ligand” and “PK modulator” refers to molecules which can modulate the pharmacokinetics of the composition of the disclosure. Some exemplary PK modulator include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, and transthyretia- binding ligands (e.g., tetraiidothyroacetic acid, 2, 4, 6-triiodophenol and flufenamic acid). Oligomeric compounds that comprise a number of phosphorothioate intersugar linkages are also known to bind to serum protein, thus short oligomeric compounds, e.g. oligonucleotides of comprising from about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), and that comprise a plurality of phosphorothioate linkages in the backbone are also amenable to the present disclosure as ligands (e.g. as PK modulating ligands). The PK modulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and/or phosphorodithioate linkages. In some embodiments, all internucleotide linkages in PK modulating oligonucleotide are phosphorothioate and/or phosphorodithioates linkages. In addition, aptamers that bind serum components (e.g. serum proteins) Docket No.: 121301-22520 ALN-511-WO are also amenable to the present disclosure as PK modulating ligands. Binding to serum components (e.g. serum proteins) can be predicted from albumin binding assays, scuh as those described in Oravcova, et al., Journal of Chromatography B (1996), 677: 1-27. When two or more ligands are present, the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties. For example, a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In another embodiment, all the ligands have different properties. The ligand or tethered ligand can be present on a monomer when said monomer is incorporated into a component of the dsRNA agent of the disclosure (e.g., a dsRNA agent of the disclosure or linker). In some embodiments, the ligand can be incorporated via coupling to a “precursor” monomer after said “precursor” monomer has been incorporated into a component of the dsRNA agent of the disclosure (e.g., a dsRNA agent of the disclosure or linker). For example, a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., monomer- linker-NH2 can be incorporated into into a component of the compounds of the disclosure (e.g., a dsRNA agent of the disclosure or linker). In a subsequent operation, i.e., after incorporation of the precursor monomer into a component of the compounds of the disclosure (e.g., a dsRNA agent of the disclosure or linker), a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer’s tether. In another example, a monomer having a chemical group suitable for taking part in Click Chemistry reaction can be incorporated e.g., an azide or alkyne terminated tether/linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having complementary chemical group, e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together. In some embodiments, ligand can be conjugated to nucleobases, sugar moieties, or internucleosidic linkages of the dsRNA agent of the disclosure. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. When a ligand is conjugated to a nucleobase, the preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a conjugate moiety, such as in an abasic residue. Internucleosidic linkages can also bear conjugate moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to Docket No.: 121301-22520 ALN-511-WO the phosphorus atom. For amine- or amide-containing internucleosidic linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom. There are numerous methods for preparing conjugates of oligonuclotides. Generally, an oligonucleotide is attached to a conjugate moiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl, aldehyde, and the like) on the oligonucleotide with a reactive group on the conjugate moiety. In some embodiments, one reactive group is electrophilic and the other is nucleophilic. For example, an electrophilic group can be a carbonyl-containing functionality and a nucleophilic group can be an amine or thiol. Methods for conjugation of nucleic acids and related oligomeric compounds with and without linking groups are well described in the literature such as, for example, in Manoharan in Antisense Research and Applications, Crooke and LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, which is incorporated herein by reference in its entirety. The ligand can be attached to the dsRNA agent of the disclosures via a linker or a carrier monomer, e.g., a ligand carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier monomer into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of an oligonucleotide. A “tethering attachment point” (TAP) in refers to an atom of the carrier monomer, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The selected moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the carrier monomer. Thus, the carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent atom. Representative U.S. patents that teach the preparation of conjugates of nucleic acids include, but are not limited to, U.S. Pat. Nos.4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578, 717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118, 802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578, 718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904, 582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082, 830; 5,112,963; 5,149,782; 5,214,136; 5,245,022; 5,254, 469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317, 098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510, 475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599, 923; 5,599,928; 5,672,662; 5,688,941; 5,714,166; 6,153, 737; 6,172,208; 6,300,319; 6,335,434; 6,335,437; 6,395, 437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; 6,559, 279; contents of which are herein incorporated in their entireties by reference. Docket No.: 121301-22520 ALN-511-WO In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a liver tissue. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the targeting ligand is a GalNAc conjugate. In certain embodiments, the dsRNA agent of the disclosure further comprises a ligand having a structure shown below:
Figure imgf000101_0002
, wherein: LG is independently for each occurrence a ligand, e.g., carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, polysaccharide; and Z’, Z”, Z”’ and Z”” are each independently for each occurrence O or S. In certain embodiments, the dsRNA agent of the disclosure comprises a ligand of Formula ,
Figure imgf000101_0003
Formula (IV) , or Formula (V) , wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; Q and Q’ are independently for each occurrence is absent, –(P7-Q7-R7)p-T7- or –T7-Q7-T7’-B-
Figure imgf000101_0001
T8 and T8’ are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O; B is –CH2-N(BL)-CH2-; BL is –TB-QB-TB’-Rx; Docket No.: 121301-22520 ALN-511-WO Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C, Q7, Q8 and QB are independently for each occurrence absent, alkylene, substituted alkylene and wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN$& 7#Ej$47#Ej$& 7m7 Z\ C(O); TB and TB’ are each independently for each occurrence absent, CO, NH, O, S, OC(O), OC(O)O, NHC(O), NHC(O)NH, NHC(O)O, CH2, CH2NH or CH2O; Rx is a lipophile (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid; R1, R2, R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C, R7 are each independently for each occurrence C C NHCH C -C -CH -NH-,
Figure imgf000102_0001
L1, L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C are each independently for each occurrence a carbohydrate, e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide; R’ and R” are each independently H, C1-C6 alkyl, OH, SH, or N(RN)2; RN is independently for each occurrence H, methyl, ethyl, propyl, isopropyl, butyl or benzyl; Ra is H or amino acid side chain; Z’, Z”, Z”’ and Z”” are each independently for each occurrence O or S; p represent independently for each occurrence 0-20. As discussed above, because the ligand can be conjugated to the iRNA agent via a linker or carrier, and because the linker or carrier can contain a branched linker, the iRNA agent can then contain multiple ligands via the same or different backbone attachment points to the carrier, or via the branched linker(s). For instance, the branchpoint of the branched linker may be a bivalent, trivalent, tetravalent, pentavalent ,or hexavalent atom, or a group presenting such multiple valencies. In certain embodiments, the branchpoint is -N, -N(Q)-C, -O-C, -S-C, -SS-C, -C(O)N(Q)-C, -OC(O)N(Q)-C, - N(Q)C(O)-C, or -N(Q)C(O)O-C; wherein Q is independently for each occurrence H or optionally substituted alkyl. In other embodiment, the branchpoint is glycerol or glycerol derivative. Docket No.: 121301-22520 ALN-511-WO Suitable ligands for use in the compositions of the disclosure are described in U.S. Patent Nos.8,106,022, 8,450,467, 8,882,895, 9,352,048, 9,370,581, 9,370,582, 9,867,882, 10,806,791, and 11,110,174, and U.S. Patent Publication Nos.2009/239814, 200/9247608, 2012/136042, 2013/178512, 2014/179761, 2015/011615, 2015/119444, 2015/119445, 2016/051691, 2016/375137, 2018/326070, 2019/099493, 2019/184018, and 2020/297853, the entire contents of each of which are incorporated herein by reference. In some embodiments, a suitable ligand is a ligand disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment the ligand comprises the structure below:
Figure imgf000103_0003
In certain embodiments, the dsRNA of the disclosure a ligand of structure:
Figure imgf000103_0001
In certain embodiments, the dsRNA agent of the disclosure is conjugated with a ligand of structure:
Figure imgf000103_0002
In certain embodiments, the dsRNA agent of the disclosure comprises a ligand of structure: Docket No.: 121301-22520 ALN-511-WO
Figure imgf000104_0001
In certain embodiments, the dsRNA agent of the disclosure comprises a monomer of structure:
Figure imgf000104_0002
. In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S Docket No.: 121301-22520 ALN-511-WO
Figure imgf000105_0001
. In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:
Figure imgf000105_0002
. Synthesis of above described ligands and monomers is described, for example, in US Patent No.8,106,022, content of which are incorporated herein by reference in their entirety. VIII. Delivery of an RNAi Agent of the Disclosure The delivery of a RNAi agent of the disclosure to 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 an ACVR1C-associated disorder, e.g., metabolic disorder, can be achieved in a number of different ways. For example, delivery 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. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These alternatives are discussed further below. In general, 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 (see e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol.2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, 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 non-specific effects of an RNAi agent can Docket No.: 121301-22520 ALN-511-WO be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the RNAi agent to be administered. Several studies have shown successful knockdown of gene products when an RNAi agent is administered locally. For example, pulmonary delivery, e.g., inhalation, of a dsRNA, e.g., SOD1, has been shown to effectively knockdown gene and protein expression in lung tissue and that there is excellent uptake of the dsRNA by the bronchioles and alveoli of the lung. Intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ. et al. (2003) Mol. Vis.9:210- 216) were also both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, WJ. et al., (2006) Mol. Ther.14:343-350; Li, S. et al., (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther.12:59-66; Makimura, H. et a.l (2002) BMC Neurosci.3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya,Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, KA. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem.279:10677-10684; Bitko, V. et al., (2005) Nat. Med.11:50-55). For administering a RNAi agent systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the RNAi agent to the target tissue and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, use of GNAs as described herein has been identified to destabilize the seed region of a dsRNA, resulting in enhanced preference of such dsRNAs for on-target effectiveness, relative to off-target effects, as such off-target effects are significantly weakened by such seed region destabilization). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, a RNAi agent directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an RNAi agent to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol.24:1005-1015). In an alternative embodiment, the RNAi agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of molecule RNAi agent (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an RNAi agent, or induced to form a Docket No.: 121301-22520 ALN-511-WO vesicle or micelle (see e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an RNAi agent. The formation of vesicles or micelles further prevents degradation of the RNAi agent when administered systemically. Methods for making and administering cationic- RNAi agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res.9:1291-1300; Arnold, AS et al. (2007) J. Hypertens.25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111- 114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther.12:321-328; Pal, A. et al., (2005) Int J. Oncol.26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm.3:472-487), and polyamidoamines (Tomalia, DA. et al., (2007) Biochem. Soc. Trans.35:61-67; Yoo, H. et al., (1999) Pharm. Res.16:1799-1804). In some embodiments, a RNAi agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Patent No.7, 427, 605, which is herein incorporated by reference in its entirety. Certain aspects of the instant disclosure relate to a method of reducing the expression of an ACVR1C gene in a cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure. In one embodiment, the cell is a liver cell. In one embodiment, the cell is an adipocyte. In certain embodiments, the RNAi agent is taken up on one or more tissue or cell types present in organs, e.g., liver, adipose tissue. Another aspect of the disclosure relates to a method of reducing the expression and/or activity of an ACVR1C 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 an ACVR1C- associated disorder or at risk of having or at risk of developing an ACVR1C-associated disorder, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the disclosure, thereby treating the subject. In some embodiments, the an ACVR1C- associated disorder comprises a metabolic disorder. In some embodiments, the metabolic disorder comprises metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight. In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In one embodiment, the double-stranded RNAi agent is administered intramuscularly. In one embodiment, the double-stranded RNAi agent is administered by intravenously. In one embodiment, the double-stranded RNAi agent is administered by pulmonary sytem administration, e.g., intranasal administration, or oral inhalative administration. Docket No.: 121301-22520 ALN-511-WO For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the disclosure. 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, intraventricular, 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 and a pharmaceutically acceptable carrier. As used herein the language “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 substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be 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, or intrathecal or intraventricular administration. 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. Compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration. Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives. Such administration permits both systemic and local delivery of the double stranded RNAi agents of the disclosure. Intranasal administration may include instilling or insufflating a double stranded RNAi agent into the nasal cavity with syringes or droppers by applying a few drops at a time or via atomization. Suitable dosage forms for intranasal administration include drops, powders, nebulized mists, and sprays. Nasal delivery devices include, but not limited to, vapor inhaler, nasal dropper, spray bottle, metered dose spray pump, gas driven spray atomizer, nebulizer, mechanical powder sprayer, breath Docket No.: 121301-22520 ALN-511-WO actuated inhaler, and insufflator. Devices for delivery deeper into the respiratory system, e.g., into the lung, include nebulizer, pressured metered-dose inhaler, dry powder inhaler, and thermal vaporization aerosol device. Devices for delivery by inhalation are available from commercial suppliers.Devices can be fixed or variable dose, single or multidose, disposable or reusable depending on, for example, the disease or disorder to be prevented or treated, the volume of the agent to be delivered, the frequency of delivery of the agent, and other considerations in the art. Oral inhalative administration may include use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI), to deliver a double stranded RNAi agent to the pulmonary system. Suitable dosage forms for oral inhalative administration include powders and solutions. Suitable devices for oral inhalative administration include nebulizers, metered-dose inhalers, and dry powder inhalers. Dry powder inhalers are of the most popular devices used to deliver drugs, especially proteins to the lungs. Exemplary commercially available dry powder inhalers include Spinhaler (Fisons Pharmaceuticals, Rochester, NY) and Rotahaler (GSK, RTP, NC). Several types of nebulizers are available, namely jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers. Jet nebulizers are driven by compressed air. Ultrasonic nebulizers use a piezoelectric transducer in order to create droplets from an open liquid reservoir. Vibrating mesh nebulizers use perforated membranes actuated by an annular piezoelement to vibrate in resonant bending mode. The holes in the membrane have a large cross-section size on the liquid supply side and a narrow cross- section size on the side from where the droplets emerge. Depending on the therapeutic application, the hole sizes and number of holes can be adjusted. Selection of a suitable device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung. Aqueous suspensions and solutions are nebulized effectively. Aerosols based on mechanically generated vibration mesh technologies also have been used successfully to deliver proteins to lungs. The amount of RNAi agent for pulmonary system administration may vary from one target gene to another target gene and the appropriate amount that has to be applied may have to be PQ^Q\XUYQP UYPU`UP_MWWc RZ\ QMOT ^M\SQ^ SQYQ( Gc[UOMWWc& ^TU] MXZ_Y^ \MYSQ] R\ZX *) tS ^Z + XS& Z\ .) tS ^Z *.)) tS& Z\ *)) tS ^Z *))) tS( Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical 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. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening or flavoring agents can be added. Compositions suitable for oral Docket No.: 121301-22520 ALN-511-WO administration of the agents of the disclosure are further described in PCT Application No. PCT/US20/33156, the entire contents of which are incorporated herein by reference. Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives. Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic. In one embodiment, the administration of the siRNA compound, e.g., a double-stranded siRNA compound, is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary system, intranasal, urethral, or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below. A. Vector encoded RNAi agents of the Disclosure RNAi agents targeting the ACVR1C 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. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating 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 cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, 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 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. Delivery of 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 Docket No.: 121301-22520 ALN-511-WO 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 or avipox, e.g. 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. Alternatively, 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 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. IX. Pharmaceutical Compositons The present disclosure also includes pharmaceutical compositions and formulations which include the RNAi agents of the disclosure. In one embodiment, provided herein are pharmaceutical compositions containing an RNAi agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the RNAi agent are useful for treating a subject who would benefit from inhibiting or reducing the expression of an ACVR1C gene , e.g., a subject having an ACVR1C-associated disorder, e.g., a subject having or at risk of having or at risk of developing an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery. In some embodiments, the pharmaceutical compositions of the disclosure are pyrogen free or non-pyrogenic. In one embodiment, 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 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. In one example the microscopic vesicles are liposomes. In some embodiments the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.In one aspect, the disclosure features 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 which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In some embodiments the sterile Docket No.: 121301-22520 ALN-511-WO solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol. In one aspect, the disclosure features 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 which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In some embodiments the enteric material 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. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein. In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol. In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate. X. Methods For Inhibiting ACVR1C Expression Another aspect of the disclosure relates to a method of reducing the expression of an ACVR1C gene in a cell, comprising contacting the cell with a dsRNA agent of the disclosure. The methods include contacting the cell with a dsRNA of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcripts of an ACVR1C gene, thereby inhibiting expression of the ACVR1C gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of a target may be determined by determining the mRNA expression level of the target gene using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of a target protein using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. In the methods of the disclosure the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible. The cell may be an extra-hepatic cell, such as a liver cell or an adipocyte. A cell suitable for treatment using the methods of the disclosure may be any cell that expresses a target gene. A cell suitable for use in the methods of the disclosure may be a mammalian Docket No.: 121301-22520 ALN-511-WO cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a rat cell, or a mouse cell. In one embodiment, the cell is a human cell, e.g., a human liver cell or a human kidney cell. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest. In certain embodiments, the RNAi agent does not include a targeting ligand. The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition. In certain embodiments, a level of inhibition, e.g., for an RNAi agent of the instant disclosure, can be assessed in cell culture conditions, e.g., wherein cells in cell culture are transfected via LipofectamineTM-mediated transfection at a concentration in the vicinity of a cell of 10 nM or less, 1 nM or less, etc. Knockdown of a given RNAi agent can be determined via comparison of pre-treated levels in cell culture versus post-treated levels in cell culture, optionally also comparing against cells treated in parallel with a scrambled or other form of control RNAi agent. Knockdown in cell culture of, e.g., 50% or more, can thereby be identified as indicative of “inhibiting” or “reducing”, “downregulating” or “suppressing”, etc. having occurred. It is expressly contemplated that assessment of targeted mRNA or encoded protein levels (and therefore an extent of “inhibiting”, etc. caused by a RNAi agent of the disclosure) can also be assessed in in vivo systems for the RNAi agents of the instant disclosure, under properly controlled conditions as described in the art. The phrase “inhibiting expression of an ACVR1C gene” or “inhibiting expression of ACVR1C,” as used herein, includes inhibition of expression of any ACVR1C gene (such as, e.g., a mouse ACVR1C gene, a rat ACVR1C gene, a monkey ACVR1C gene, or a human ACVR1C gene) as well as variants or mutants of an ACVR1C gene that encodes an ACVR1C protein. Thus, the ACVR1C gene may be a wild-type ACVR1C gene, a mutant ACVR1C gene, or a transgenic ACVR1C gene in the context of a genetically manipulated cell, group of cells, or organism. “Inhibiting expression of an ACVR1C gene” includes any level of inhibition of an ACVR1C gene, e.g., at least partial suppression of the expression of an ACVR1C gene, such as an inhibition by at least 20%. In certain embodiments, inhibition is by at least 30%, at least 40%, at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to below the level of detection of the assay method. The expression of an ACVR1C gene may be assessed based on the level of any variable associated with ACVR1C gene expression, e.g., ACVR1C mRNA level or ACVR1C protein level. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control). Docket No.: 121301-22520 ALN-511-WO In some embodiments of the methods of the disclosure, expression of an ACVR1C gene is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of ACVR1C, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of an ACVR1C gene. Inhibition of the expression of an ACVR1C gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which an ACVR1C gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a RNAi agent of the disclosure, or by administering a RNAi agent of the disclosure to a subject in which the cells are or were present) such that the expression of an ACVR1C gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with a RNAi agent or not treated with a RNAi agent targeted to the genome of interest). The degree of inhibition may be expressed in terms of: (mRNAincontrolcells) - (mRNAin treated cells) ^100 % (mRNAincontrol cells) In other embodiments, inhibition of the expression of an ACVR1C gene may be assessed in terms of a reduction of a parameter that is functionally linked to an ACVR1C gene expression, e.g., ACVR1C protein expression. ACVR1C gene silencing may be determined in any cell expressing an ACVR1C gene, either endogenous or heterologous from an expression construct, and by any assay known in the art. Inhibition of the expression of an ACVR1C protein may be manifested by a reduction in the level of the ACVR1C protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of genome suppression, the inhibiton of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells. A control cell or group of cells that may be used to assess the inhibition of the expression of an ACVR1C gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the disclosure. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent. The level of ACVR1C mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing RNA expression. In one embodiment, the level of expression of ACVR1C in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the ACVR1C gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasyTM RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on Docket No.: 121301-22520 ALN-511-WO assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating ACVR1C mRNA may be detected using methods the described in WO2012/177906, the entire contents of which are hereby incorporated herein by reference. In some embodiments, the level of expression of ACVR1C is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific ACVR1C nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to ACVR1C RNA. In one embodiment, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known RNA detection methods for use in determining the level of ACVR1C mRNA. An alternative method for determining the level of expression of ACVR1C in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, US Patent No.4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., US Patent No.5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the disclosure, the level of expression of ACVR1C is determined by quantitative fluorogenic RT-PCR (i.e., the TaqManTM System), by a Dual-Glo® Luciferase assay, or by other art-recognized method for measurement of ACVR1C expression or mRNA level. The expression level of ACVR1C mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See US Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of ACVR1C expression level may also comprise using nucleic acid probes in solution. Docket No.: 121301-22520 ALN-511-WO In some embodiments, the level of RNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of ACVR1C nucleic acids. The level of ACVR1C protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme- linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the detection of proteins indicative of the presence or replication of ACVR1C proteins. In some embodiments, the efficacy of the methods of the disclosure in the treatment of an ACVR1C-related disease is assessed by a decrease in ACVR1C mRNA level (e.g, by assessment of a blood ACVR1C gene level, or otherwise). In some embodiments, the efficacy of the methods of the disclosure in the treatment of an ACVR1C-related disease is assessed by a decrease in ACVR1C mRNA level (e.g, by assessment of a liver or kidney sample for ACVR1C level, by biopsy, or otherwise). In some embodiments of the methods of the disclosure, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of ACVR1C may be assessed using measurements of the level or change in the level of ACVR1C mRNA or ACVR1C protein in a sample derived from a specific site within the subject, e.g., liver or kidney cells. In certain embodiments, the methods include a clinically relevant inhibition of expression of ACVR1C, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of ACVR1C. As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used. XI. Prophylactic and Treatment Methods of the Disclosure The present disclosure also provides methods of using an iRNA of the disclosure or a composition containing an iRNA of the disclosure to inhibit expression of ACVR1C, thereby preventing or treating an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight. In the methods of the disclosure the cell may be contacted with the siRNA in vitro or in vivo, i.e., the cell may be within a subject. Docket No.: 121301-22520 ALN-511-WO A cell suitable for treatment using the methods of the disclosure may be any cell that expresses an ACVR1C gene, e.g., an adipocyte cell, or a liver cell. A cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell, including human cell in a chimeric non-human animal, or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), or a non-primate cell. In certain embodiments, the cell is a human cell, e.g., a human liver cell. In the methods of the disclosure, ACVR1C gene expression is inhibited in the cell by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or to a level below the level of detection of the assay. The in vivo methods of the disclosure may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ACVR1C gene of the mammal to which the RNAi agent is to be administered. The composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, intraocular (e.g., periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, anterior or posterior juxtascleral, subretinal, subconjunctival, retrobulbar, or intracanalicular injection), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by intramuscular injection. The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting. In one aspect, the present disclosure also provides methods for inhibiting the expression of an ACVR1C gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an ACVR1C gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the ACVR1C gene, thereby inhibiting expression of the ACVR1C gene in the cell. Reduction in gene expression can be assessed by any methods known in the art and by methods, e.g. qRT-PCR, described herein, e.g., in Example 2. Reduction in protein production can be assessed by any methods known it the art, e.g. ELISA. In certain embodiments, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the target gene or protein expression. In other embodiments, a blood sample serves as the subject sample for monitoring the reduction in the target protein expression. The present disclosure further provides methods of treatment in a subject in need thereof, e.g., a subject diagnosed with an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, a disorder of body weight. Docket No.: 121301-22520 ALN-511-WO The present disclosure further provides methods of prophylaxis in a subject in need thereof. The treatment methods of the disclosure include administering an iRNA of the disclosure to a subject, e.g., a subject that would benefit from a reduction of expression of an ACVR1C gene, in a prophylactically effective amount of a dsRNA targeting ACVR1C or a pharmaceutical composition comprising a dsRNA targeting ACVR1C. In one aspect, the present disclosure provides methods of treating a subject having a disorder that would benefit from reduction in expression of an ACVR1C gene, e.g., an ACVR1C-associated disorder, e.g., a metabolic disorder, e.g., diabetes. Treatment of a subject that would benefit from a reduction and/or inhibition of ACVR1C gene expression includes therapeutic treatment (e.g., a subject is having an ACVR1C-associated disorder, e.g., a metabolic disorder) and prophylactic treatment (e.g., the subject is not having an ACVR1C-associated disorder, or a subject may be at risk of developing an ACVR1C-associated disorder). In some embodiments, the an ACVR1C-associated disorder is a metabolic disorder. Examples of metablic disorder include but are not limited to, metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight. In some embodiments, the metablic disorder is metabolic syndrome. In some embodiments, the RNAi agent is administered to a subject in an amount effective to inhibit expression of ab activin A receptor type 1C (ACVR1C) gene in a cell within the subject. The amount effective to inhibit ACVR1C expression in a cell within a subject may be assessed using methods discussed above, including methods that involve assessment of the inhibition of ACVR1C mRNA, ACVR1C protein, or related variables, such as insulin resistance, BMI, WHRadj BMI, adipose tissue, e.g., image-based quantification of adipose tissue, e.g., MRI or DEXA for abdominal subcutaneous adipose and visceral adipose tissue quantification. An iRNA of the disclosure may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject. Alternatively, an iRNA of the disclosure may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation. Subjects that would benefit from an inhibition of ACVR1C gene expression are subjects susceptible to or diagnosed with an ACVR1C-associated disorder, e.g., a metablic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight. In an embodiment, the method includes administering a composition featured herein such that expression of the target gene is decreased, such Docket No.: 121301-22520 ALN-511-WO as for about 1, 2, 3, 4, 5, 6, 1-6, 1-3, or 3-6 months per dose. In certain embodiments, the composition is administered once every 3-6 months. In one embodiment, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the ACVR1C gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein. Administration of the iRNA according to the methods of the disclosure may result prevention or treatment of an ACVR1C-associated disorder, e.g., a metablic disorder, e.g., metabolic syndrome, a disorder of carbohydrates, e.g., type II diabetes, pre-diabetes, a lipid metabolism disorder, e.g., a hyperlipidemia, hypertension, lipodystrophy; a kidney disease; a cardiovascular disease, or a disorder of body weight. Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg. In one embodiment, the iRNA is administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired dose of iRNA to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as once per month to once a year. In certain embodiments, the iRNA is administered about once per month to about once every three months, or about once every three months to about once every six months. The disclosure further provides methods and uses of an iRNA agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction and/or inhibition of ACVR1C gene expression, e.g., a subject having an ACVR1C-associated disorder, e.g., a metabolic disorder, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. Accordingly, in some aspects of the disclosure, the methods which include administration of an iRNA agent of the disclosure, further include administering to the subject one or more additional therapeutic agents. For example, in certain embodiments, a dsRNA targeting ACVR1C is administered in combination with, e.g., an agent useful in treating an ACVR1C-associated disorder, e.g., a metabolic disorder, as described herein or otherwise known in the art. For example, additional agents and treatments suitable for treating a subject that would benefit from reducton in ACVR1C expression, e.g., a subject having an ACVR1C-associated disorder, e.g., a metabolic disorder, may include agents currently used to treat symptoms of an ACVR1C-associated disorder. Examples of the additional therapeutic agents which can be used with a dsRNA agent of the disclosure include, but are not limited to, insulin, a glucagon-like peptide 1 agonist (e.g., exenatide, liraglutide, dulaglutide, semaglutide, and pramlintide), a sulfonylurea (e.g., chlorpropamide, glipizide), a seglitinide (e.g., repaglinide, nateglinidie), biguanides (e.g., metformin), a Docket No.: 121301-22520 ALN-511-WO thiazolidinedione, e.g, rosiglitazone, troglitazone, an alpha-glucosidase inhibitor (e.g., acarbose and meglitol ), an SGLT2 inhibitor (e.g., dapagliflozin), a DPP-4 inhibitor (e.g., linagliptin), or an HMG- CoA reductase inhibitor, e.g., statins, such as atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor), lovastatin extended-release (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin (Zocor). Additional therapeutic agents can also include other siRNAs targeting the liver (e.g. INHBE). The iRNA agent and an additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein. XII. Kits In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a siRNA compound, or a DNA which encodes an siRNA compound, e.g., a double- stranded siRNA compound, or ssiRNA compound, or precursor thereof). Such kits include one or more dsRNA agent(s) and instructions for use, e.g., instructions for administering a prophylactically or therapeutically effective amount of a dsRNA agent(s). The dsRNA agent may be in a vial or a pre-filled syringe. The kits may optionally further comprise means for administering the dsRNA agent (e.g., an injection device, such as a pre-filled syringe), or means for measuring the inhibition of an ACVR1C gene (e.g., means for measuring the inhibition of ACVR1C mRNA, ACVR1C protein, and/or ACVR1C activity). Such means for measuring the inhibition of ACVR1C gene may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the disclosure may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount. In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container, e.g., a vial or a pre-filled syringe. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device. This disclosure is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the informal Sequence Listing and Figures, are hereby incorporated herein by reference. Docket No.: 121301-22520 ALN-511-WO EXAMPLES Example 1. siRNA Design and Synthesis Source of reagents Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology. siRNA Design siRNAs targeting the activin A receptor type 1C (ACVR1C) gene (ACVR1C, human: NCBI refseqID NM_145259.3, NCBI Gene ID: 130399) were designed using custom R and Python scripts. The human NM_145259.3 mRNA has a length of 8853 bases. Detailed lists of the unmodified sense and antisense strand sequences of ACVR1C dsRNA agents are shown in Table 2. Detailed lists of the modified sense and antisense strand sequences of ACVR1C dsRNA agents are shown in Table 3. Detailed lists of the unmodified sense and antisense strand sequences of ACVR1C dsRNA agents comprising an unsaturated C22 hydrocarbon chain conjugated to position 6 on the sense strand, counting from the 5’-end of the sense strand are shown in Table 4. Detailed lists of the modified sense and antisense strand sequences of ACVR1C dsRNA agents comprising an unsaturated C22 hydrocarbon chain conjugated to position 6 on the sense strand, counting from the 5’-end of the sense strand are shown in Table 5. It is to be understood that, throughout the application, a duplex name without a decimal is equivalent to a duplex name with a decimal which merely references the batch number of the duplex. For example, AD-2410459 is equivalent to AD-2410459.1. siRNA Synthesis siRNAs were designed, synthesized, and prepared using methods known in the art. Briefly, as an exemplary method, siRNA sequences were synthesized on a 1 µmol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on solid supports. Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramidite monomers (2’- deoxy-2’-fluoro, 2’-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, WI), Hongene (China), or Chemgenes (Wilmington, MA, USA). Additional phosphoramidite monomers were procured from commercial suppliers, prepared in-house, or procured using custom synthesis from various CMOs. Phosphoramidites were prepared at a concentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMF and were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) with a reaction time of 400 s. The lipophilic moiety (e.g., the exemplary C22 conjugate) were introduced as the lipophilic monomer phosphoramidites. Synthesis of C22-nucleoside phosphoramidites for the synthesis of dsRNA agent conjugates is described in WO2023/064530. Docket No.: 121301-22520 ALN-511-WO Phosphorothioate linkages were generated using a 100 mM solution of 3-((Dimethylamino- methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, MA, USA)) in anhydrous acetonitrile/pyridine (9:1 v/v). Oxidation time was 5 minutes. All sequences were synthesized with final removal of the DMT group (“DMT-Off”). Upon completion of the solid phase synthesis, solid-supported oligoribonucleotides were treated with 300 µL of Methylamine (40% aqueous) at room temperature in 96 well plates for approximately 2 hours to afford cleavage from the solid support and subsequent removal of all additional base-labile protecting groups. For sequences containing any natural ribonucleotide linkages (2’-OH) protected with a tert-butyl dimethyl silyl (TBDMS) group, a second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride). To each oligonucleotide solution in aqueous methylamine was added 200 µL of dimethyl sulfoxide (DMSO) and 300 µL TEA.3HF and the solution was incubated for approximately 30 mins at 60 °C. After incubation, the plate was allowed to come to room temperature and crude oligonucleotides were precipitated by the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1 ethanol:isopropanol. The plates were then centrifuged at 4 °C for 45 mins and the supernatant carefully decanted with the aid of a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5 mL, GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector. Desalted samples were collected in 96 well plates and then analyzed by LC-MS and UV spectrometry to confirm identity and quantify the amount of material, respectively. Duplexing of single strands was performed on a Tecan liquid handling robot. Sense and antisense single strands were combined in an equimolar ratio to a final concentration of 10 µM in 1x PBS in 96 well plates, the plate sealed, incubated at 100 °C for 10 minutes, and subsequently allowed to return slowly to room temperature over a period of 2-3 hours. The concentration and identity of each duplex was confirmed and then subsequently utilized for in vitro screening assays. Example 2. In vitro screening methods Cell culture and 384-well transfections SK-MEL-28 cells (Cat # HTB-72, ATCC, Manassas, VA) were grown to near confluence at 37°C in an atmosphere of 5% CO2 in Eagle’s Minimum Essential Medium (Gibco) supplemented with 10% FBS (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 5 ql of Opti-MEM plus 0.1 ql of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA. cat # 13778-150) to 5 ql of each siRNA duplex to an individual well in a 384-well plate. The mixture was then incubated at room temperature for 15 minutes. Forty ql of complete growth media without antibiotic containing ~5 x103 cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM, 1 nM, and 0.1 nM final duplex concentration. Total RNA isolation using DYNABEADS mRNA Isolation Kit (Invitrogen™, part #: 610-12) Docket No.: 121301-22520 ALN-511-WO RNA was isolated using an automated protocol on a BioTek-EL406 platform using Dynabeads™ mRNA DIRECT™ Purification Kit (Invitrogen™, Catalog No.61012). Briefly, 70 µL of Lysis/Binding Buffer and 10 µL of lysis buffer containing 3 µL of magnetic beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room temperature and then magnetic beads were captured and the supernatant was removed. Bead-bound RNA was then washed 2 times with 90 µL Wash Buffer A and once with 90 µL Wash Buffer B. Beads were then washed with 90 µL Elution Buffer, re-captured, and supernatant was removed. cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, Cat #4374967) Complementary DNA (cDNA) was synthesized using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems™, Catalog No.4374967) according to the manufacturer’s recommendations. A master mix containing 1 µL 10X Buffer, 0.4 µL 25X deoxyribonucleotide triphosphate, 1 µL 10X Random primers, 0.5 µL Reverse Transcriptase, 0.5 µL RNase inhibitor, and 6.6 µL of water per reaction was added to RNA isolated above. The plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, followed by 2 hours incubation at 37°C Real time PCR Two microlitre (µL) of cDNA were added to a master mix containing 0.5µL of human GAPDH TaqMan Probe (4326317E), 0.5µL human ACVR1C (Taqman ID Hs00899854_m1), 2µL nuclease-free water and 5µL Lightcycler 480 probe master mix (Roche Cat # 04887301001) per well in a 384 well plates (Roche cat # 04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche). GZ OMWO_WM^Q \QWM^U`Q RZWP OTMYSQ& PM^M aQ\Q MYMWcdQP _]UYS ^TQ pp7^ XQ^TZP MYP YZ\XMWUdQP to assays performed with cells transfected with 10nM AD-1955, or mock transfected cells. The sense and antisense sequences of AD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14964) and antisense UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 14965). Table 6 shows the results of three dose screens in SK-MEL-28 cells transfected with the indicated agents from Tables 2 and 3. Table 1. Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds; and it is understood that when the nucleotide contains a 2’- fluoro modification, then the fluoro replaces the hydroxy at that position in the parent nucleotide (i.e., it is a 2’-deoxy-2’-fluoronucleotide). It is to be further understood that the nucleotide abbreviations in the table omit the 3’-phosphate (i.e., they are 3’-OH) when placed at the 3’-terminal position of an oligonucleotide.
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S d ' A A A A A A A A A A A A A A n 3 A A A A A A A A G U G U G A a r A A A A U A U G U C U U U U t o t A G G G U G U C G G U C U U G U G U G G G U G U C G G U A U G U U G U C U U U G U G U S '5 AA A G G G G U G U C G G U A U U U G U C U U U U G U G es A A A A A A G G G U G U C U G U A U A U G U C U U U U G U n e c C A A A A G G G U G U G U G G U G U A U G U C U U U U G es n U C A A A A G G G U G G U U U G U G U A U G U C U U U U it eu U U C A A A A G G G G GA G U G U G U A U G U C U U U n q C U U C AC A A A A A G G G A A G U G U G U A U G U C U U A e A S C C U U A A U C A A A A G G A A G U G U G U A U G U C UC dn d G A C C CA UC U U C A A A A U G A A G U G U G U A U G U G G C A C U U C A A A C U G A A G U G U G U A U U a na U A U G es r G C G C A C U U C A A A C U G A A G U G G U t C G C G C A C U U C A G A C U G A A G U U A U G A G U G G U G U G U A n S C C G C G C A C U U C G G A C eS e s A A C C G C G C A A UC U G AA G U G U G U C C UC U A G G A G A C U G A A G U G U G n C A C C d C A A C GC C GC G AC A U CC A G G A C G A A U G U C A A C GC G C C C CC A G G G A UC G A GA G U G e e i S G C C A A C C G G C G A C G C G C C A C A G G A U C U G A A G U fi do e 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m m 82 92 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 n a U 4 4 34 34 34 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 N 81 81 81 81 8 4 1 8 4 1 8 4 1 8 4 1 8 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 81 81 81 81 8 8 8 8 8 8 8 8 8 8 8 8 8 . x 4 4 4 4 4 4 4 4 4 4 4 4 4 14 14 14 1 1 1 1 1 1 1 1 1 1 2 e l el p 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 2 4 4 4 4 4 4 4 4 u - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 2 2 D - D - - b D D a D A A A A A A A A A A A A A A A A A A A A A A A A A A T Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' A A 3 U U AC A A A U U C AA AA A A A A A A A A A A A A A A A A A A A A ot A U U C U U C A A C A A UC U U G A A G A A U G C U C A A G U ' G A U U C U U C A A A UC U U A C U A G A U G C U C A A G 5 U G A U U C U U C A A A G U UC CA A A A AA A U G C U C A A ec G U G A U G U G U U C A U U UC U C A n U U C A U G A A U G U A A G A U CC G A C A A U G C UC C A e G U G U G A U U C U U C C A A C A U A U G A A U G U C U G C u U G U G U G A U U C A G A U G A A A U G UC q U U G U G U G A U U UC U G C G C G A A G G C U G A A A U G e U U U G U G U G A U U A C G A U U U C U U A C A A U G G A A U S U A U U U G G G A C C A G C U G A A C U G A A d C U U U G U G U G G G A U A G A C G A A G A G A A C U G A n U UC U U U U G U a U G U U G A A A C U U A C A C A G A A C U G rt G U UC U U G U U G U G U G G A U G A U C C C C A G A A C U U G U UC U A U U U G U G U A G C A C C A G U C C A G A A C S A U G U UC e U U U U G U G C A G C U G A U A U U C C A G A A s U A U G G U A U C U U U U G U A C G UA G A UC G C U U C CC AC G A U G U C U U U U G A A G C AC C G A CC CC U U C A G n U G U A U G U C U U U U A A A C A U G U U U A C C U U U CC A e S G U G U A U G U C U U U C C U G A G A A U C U C C C C U C U U C C e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 5 5 5 75 85 95 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 4 4 4 4 4 4 64 64 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 8 8 N 8 8 8 8 8 8 8 8 48 48 48 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A A A A U AC A A AA A A A U AC AC AA AA A A A A A A A A A A ot C U U C U G G U AC UA G U U U C C A CA A A U G U U A C C ' U C U U C U U A C U G U C A G G U C C A C AC A U G U U A C 5 G U C U U U A U A U C G U C A A G U U C C A A AC A U G U U A e A G U C UC U U U U C U G U C A U A G U U C C A A A A U G U U c A A G U n A U U C C C A A U G U C A C U U C C A U G U C A U G A A C A G U A U C U U C U G U C A U A G U U C C A A AC A U G eu UC U C G A A G U C U C U G U C A U A G U U C C A A AC A U qe G C U C A A G U UC U U C U G U C A U A G U U C C A A AC AA S U G C U C A A G U C U U C U G U C AC U A G U U C U C A A C d A U G C U C A n A A U G C U C A G U C U U C U G U U AC U A G U C U CC A A C A A G U C U U C U G C G U A U A C A G U G C A a G A A U G U rt U G A A U G C C A A G U C U U UC S C G UC C A U G U C UA A A U U C C G C C AA G U C U U U C G U C U U U A U U e A U G A A U C A U G UC C A G U C UC U UC G U AC A U G U U s A A UC G A A A G UC U AC A G U UC U U UC U G U C A A G G n G A A U G e A C U G A U G AA U G C G U A C C AA G U C A G U C U U U C U G U C UA A U A S A G A C C A G A A U A U A G C U C G U C U C U G A A U G U C U A C A A G U U C U U U C U G U A C A e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 8 8 8 58 68 78 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 4 4 4 4 4 4 84 84 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 N 8 8 8 8 8 8 8 8 48 48 48 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000130_0001
' 3 A A A A A A ot A A A ' C A A A A A A A A A A A A A C A A A A A A A A A U U G C A A A A C C G A A U G G UC G C U U C A C A G A U U U G C 5 C e A CC A A A C C G A A U C A G U G C U U C AC CA AC G A U U U G c U A C A A A C CC G A A U C A U G C U U U C A AC G U U U U n U U A C A A A C A e G U U A C A A A CC G AA UA G C U G C UC U U C A A U U U U C AC A A A CC GC A A A A C G A U G C U G C C U U C C A U U U G A U G C U G G C U U A G A U U uq U G U U A C C A A A A C C A G AA U G C UC U G C U C A G A U e A U G U U A C C A A A A C C G AA U G A U G C U G C U C A G A S A A U G U U A C C A A A A C C G A A U G C U G U AC C A A G d C A A U G U U A C A CC A A A A C C G A A U G C U C U C C A n AA C A A U G U U A CC AC AA A A C C G A A U G C G U AC C ar t C A C A A U G U U S C A U C A U A C C A A A C C G A A U G U U C C U U AC C A C A A U G U A U U A C C A A A C C G A A U G G C U e C A s U U C A C A A U G A C A C A A U G U U A C C A A A C C G A A U G U U A C A U G C U C A A A CA CC G A UA C U G C n G e A U U C AC A C A S G U U C A A C AA UA U G U U A C A A A A A CC G A G C U G U A G U U C C A A C A U G U U A CC A A A A A CC U G C C A A A C A A U G U U A C C A C A A A A G C A U G U C e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 1 1 1 31 41 51 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 5 5 5 5 5 5 15 15 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 N 8 8 8 8 8 8 8 8 58 58 58 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A A ot A ' A CA A A A C A C A A AA A AC A A A A A A A A A A A A A A A A A A A AC U A A AC CA U G C A C C U U C C A A C A G C 5 C G AC A C A A AC A AC U A A A U A A C C U G C A C C U U C C A A C A G C A U A A AC CA UC G C A C C U U C C A A C A ec U G C A A C A A C A U A A C A U G C A C C U U C CC AC A C n U U G C A A C A A C A U A A C CA U G C AC C CC U U U C A A e U U U G C A A C A A C A u U U U U G C A A C A A C U q A A U AA C C U G A A A C A CC U U CC A C C U G C U C G C A C C U U C e U U U U U G C A A C A A C A U A A AC CA UC U G C A C C U U S A U U U U U G C AC A A C A A C A U A A C A C U G C A C C U d G A U U U U U G A C U G C A A A A C A U A A C A C U G C A C C n A G A U U U U C U U G C A A AC A C A U A A C A C U G C A C a CA AC G A U U U U U U G C A A AC A C A U A A C A C U G C A rt C A AC G A U U U U U U G C A A AC A C A U A A C A C U G C S U C A AC G A U U U U U G C A A A A C A U A AA C AC C U G es UC U C A A U U C C G A U A A A U U U U U G C A CA A A C A U A A AC C U C G A U U U U U G C A C AC A C AC U A A A AC C ne G C U C A G A U U A A A A U A S U G U C G U U U G C A C A C U A A C C U U A C A A C A G A U U U U U G A C A A A C A A A C A U A A e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 3 3 4 14 24 34 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 5 5 5 5 5 5 45 45 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 N 8 8 8 8 8 8 8 8 58 58 58 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000132_0001
' 3 A A A A A A ot A A A A A ' A U C A C AC A A A A A A A A A A A A A U G C C U G U C U U A A U A A A A A A G U G G U C AC A A A A C G A U 5 C A U C A G C A U C CA CC A A A C A A UA G C U G U C A A U U U C C U A C G G G U A A G A U G A A C U A A A G A A U G A ec A G C AC U C A C CC A A A U U U A U U U G A G A A G A G A n C AC G G AC U C A A CC A A A U U U C C U U C A G A A A U U e AA A AC A A U C C A CC A A A A U A C U C A A A A A C C G u C A A C G C A AC U A U C A C A C U A U G G C G G A G A G U G qe C C A A C G C C A G C A U C A C U U U U U U U U G G A A U A U S U C C A A C A G C A U C A A A U A G CC CC C A G A A G A C C C C C C A U U G C A G A C U d U U A A C A G A U C C G G G n C U U C C A A C A G C A U A a C C G U C U CA U U U A A C G A G G U A r U U C C A A C A G C A t A C C G AC U A U A C U G U A G C A U G C S C AC C UC U C C A A C A G C U C A C U U C C C U U U C C UC C G C A G C AC AA C A G C G C U A C G U C A G U U es G A U G C A CC U U C C A C A G G G AC U A G U C U U AC C G G n U C C U U C A A C A C U C U U C G e G AC C C U U U CC A A G UC G G U U C C G C C A C C A S A C C A U C U G A C C A C C U C U U C C A C G G U U C G U G G C G A C A G A G U U C U C A C U C U e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 6 6 6 96 07 17 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 5 5 5 5 5 5 75 75 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 9 N 8 8 8 8 8 8 8 8 58 58 58 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000133_0001
' 3 A A A A A A A A A A A A A A A A A A A t G AA U G U G A C A A A A A A A A o A ' A C A C U G A U U U A U U G A U G U G A A A A G U C U G A U U U A A A A A C U U U A A U G A U G U G C A G A AA A A G 5 G A A A A U C U G A U U U U A U G A U G U U AC G G C A C A G ec G A A G A U C U G A U U n U A A A A U U A U G A U G U U G A A A C G U A G A G A U C U G UA U U U A U G A U G U A A G A U AC e C G A A A G A U C U G C u G G UC G A A U G A U U U A U G A G G C A G G A G A G A U A U C U G A U U U A U G U G U C A G A qe U U U U G A AA G A G A U C U G A U U U A U U U U U A A C U U S A C A G C C U G A A A G A U C U G A U U U A G U G U AC AA A A dn A U A U C U G A A A G A U C UC G A U U U U U G G G U C G A U A AA C U C U G A A A G A U U G A U U C U U G U U GA C a A G A A A C U C U G A A A G A A UA C U G A A U C U U G U A A rt A A A A A C U C U G A A A G G U C U G C G CC U G U G G A G S G e U U G A A A C U C U G A A A A A A U UC s C G G A A A U A CC U G U G C G A A C U C U G G A A GA A UA U C U A C U U U U A n G A A A C U C U A G C U C A U G U A e S A C G G A A C U G A A U U G U U A A AA AA CA UC C G A AA GA G A U C CC U G G C A U C G U G G A A A A A U C U U C U G A A A G G U A C C U C U G U e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 9 9 9 79 89 99 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 5 5 5 5 5 5 06 06 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 N 8 8 8 8 8 8 8 8 68 68 68 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A A A U U A t U AA A A A A A A U U AA A A A A A A A A A AA A A A o U U G A ' U G A U A U C U U G G U A U A U C U U G U U G U U U U G U U U U G G AA A A G U C U A C U U G U U A UC U C A A 5 A U A A A G A U C UC U G G U U C C C U G G U C C U G C A A A ec G n A U A A A A G A U U G A A A A U U A C U G U A A U C C U G G U U G G A C A A G A G A U U U U A A AC U C U U G U U U U C A e U A U G A A u G G G U G G A A A G A C U G A A A A C U CC U U G A C C A C G AA A A G U C G G G A A AC CC U G C G G G A q G A U G U UA U G A A A A U U A U A A A UA C U C U U U U U G eS AA U C U G G A U G A A G A U C G A A A A C A U U U U C G C U A G G C UC A G A U G A A G C G C G A A A C A C U AC U G C dn A G G G G G G A G A U G A A U A A U G A A A C C G U U UC G ar t G A U S G A G A G A G A U A AA A G GC A U G A A A U G U A G UC A A G U A U G U C A G A G A G G G U G C AC U A A A A U U C U G es U G A G C C A G A G U A A U A G A A A G A AA C U G U U U G G G U A A U U C C A G A A C G A A C UC GC G CA U G AA C G U A C UC C CC AC G A G G U G G C G AC U G A C U C U ne A A A G C U C A A U A U U U A S A G U U G A A U C U C C U G U U G C A C A G U C C U C C A G A U A U C U G G A A C G A C U C G U U e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 2 2 2 52 62 72 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 6 6 6 6 6 6 26 26 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 N 8 8 8 8 8 8 8 8 68 68 68 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A A A A A A A A A U U C A AA A A A A A A A A A A AA A G U A A A ot U G G A C G A A U C A AC A U U A U U U G A A U C G A A A G ' A U G A A U A U A U G A U A C U A U U U G G A U G GA UC U 5 G A U G A U G A U A U A A U A C U A C U U U U A U G A U A G G e A A A G G C U A A U A G U A G A U U G A C U A U U U A A C A A c A U A U G A n C G A A U U U G U A A C U A G A C U A U U A U A C e C A U A U G U U G A U A CC U U C A U G A C U A U U G A C G u A G q A A A A G A C U G A C U U C A U A U G A C U A U G G U U e A A G U A A A U U U U C U U U C U U A U A U G A C U U U U A U S CA A A G A A G A U UA C A A G C U U A U A U G A C A C A G A d G C A A G A G G U A U A G U G G C U U A U A U G A UC G A U A n A C A A U UC G G U U A G G C C U U A U A U G A G G A A C a UC AC A A A A A G U G G C G A G U C C U U A U A U G U G G U rt G A AC C A G G C U G U C A C U C CC U U A U A U G UC G C S e U G A A C A G C U U A C A CA G C UC U CC U U A U A CC U s C A A A U G A G G C U U A G U C U G C G C U U A G C U C U A G C n G C e U G U A A C G C A U U G G G A G A C U U U U G G G A G G G C U CC UC U U A G U G GC S A C G G U U C G G G C C U U G G U U G U G A A U U U U G A C A A A C A G G G U C U C C U G G C U G e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 5 5 5 35 45 55 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 6 6 6 6 6 6 56 56 5 5 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 N 8 8 8 8 8 8 8 8 68 68 68 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000136_0001
' 3 A A A A A A A A A A A A A G U A AC AA AC A A t A A A A A A A A A A A A o U A G A U U G U U G G G A ' C G A G A U U G U U G G U A C A C A A G A U U A U U G C U U G G U A C A C A A U C C U A U U G 5 U G G G A G A U U G U G U U G G U A C A C A G CA G C U A U U ec G U G G A G A U n U A U G A U U G U U G G U A C A C G A U G C U A UA A G G A A G A U U G U U G G U A C A A A CC U e G U C G C UC uq A AC U U G G G A G A U U G U U G G U A C A U A C U G U G C A A U G G G A G A U U G U U G G U A C G A A CC UC U G e U S C U C U A A U U A U G G A G A U U G U U G G U A G A A A C C U U U C A U A U G G A G A U U G U U G G C U G A A U A A A C C d G C U U C A U A U G G A G A U U G U A G U U U A G U A A A C n U C C U U C A U A U G G A G U U C G A U G U G A G G U A A A a U A C C U U C A U A U G G A U U G A G A U U G G C A G G U A A rt A C A A C C C U U C A U A A G U G A G A U U U A A A G G U A S A C A AC C C U U C A U U A G G G A G A U U U C A A A G G U e C G C A AC C C U U C AC A U U A U G G A G A G G C C A A G G s UC G G C A AC CA C U U U C A A U G G A G U G A AC C A A G ne G G C C C U U C U A G A C A A S U C U C U G G A C C A A C U U G A U U U A C C C A C C U C U U A C A U A U G G A U G U A A C A A C e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 7 7 8 18 28 38 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 6 6 6 6 6 6 86 86 8 8 8 8 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 N 8 8 8 8 8 8 8 8 68 68 68 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000137_0001
' 3 A A A A A A ot U C ' C A A A A A A AC AA A A AA A A A A A A A A A A A A G U U C U C A G A G A A C U A A A A A U C A A A G A A U A U G 5 G C U C U C G A G G A C U U AA AA A U C A A A G A A U G U e U G C U AC U C G A C A G A AC A U A A A U C A A A G A A A G c U U G C U AC U G n A U U G C A U C G A G A C A U AA A A U C A A A G A A A U C A U C G A G A C A A U A A A U C AC A A G A A eu U A U U G C C U C A U C G A G A C A A AA A U U A U U G C U C A U C G A G A C U A A U AC A A A A A A A A A A A C A A A q G C U A U U G C U C A U C G A G A C U U A A U A A A A U C G A eS U G C U A U U G C U C C U A U C G A G A C A U A A A A U U G C G C G C A U C G A C A A G U d U U A U U G C U A G A A U A A n C a A CC U G C U A U U C U G C C U C G A G A C A U A A A A GA rt A S A A C A C U G C U A U U G UC U AC U A C A C G A G A C A U A A U U C U G C U A U U U G C U C U C G A G A CA A U A UC e U A A s A A CC U G C U A C U G C U A A U U G C UC C U C G A U C A G C A G A G A C U U A U C G U A A C U A U G U C A A C A U n G G U A A C C U G G C U UA U U G C U AC UA C GC G G G A A e S A G G U A AA CA CC U A A G G U A A A C G C A U U G C C U A U C A G UA U C U C U G U C U A U U G U C U A C A U G C G A A A e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 0 0 0 90 01 11 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 7 7 7 7 7 7 17 17 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 N 8 8 8 8 8 8 8 8 78 78 78 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000138_0001
' 3 A G A A A A A A A A AC A A G U G AC A A A A A A A A A A A A A A A A A ot U G A AA A UC U C U G G C U A U A U G A U A U C U U U G A ' G U G A A A U U C U G AC UA AA U C U C C U A A G C A U G 5 U G U G A A A UC G U A A C U U C C C A A A A C C U A G G A U ec GA U G U G A A A C U G G G C U C UA A C U A AC A A A U U A G A G U G U G A A A UC U U U G U U A A G A A G C A A UC U G n A A G U G U G A A A UC G U A U C A UC G A C A U A G UC U eu A A A G U G U G A A A UC U G G A U C A U G U G C G U G UC q A A A e A G U G U G A A A U U U G U U U C U A C C U U U U A U G S A G AA AA A A A A G A U G U G A A C U A U G U A A C C U A G UA A A CA C A A CC A A U d U G A A G G A A A A U A U A A A A n G AA A AA A G U G U G A A G C U G C A U C C G UC G A A a G A U U A A A A G U G U A G A A G AC U U AC A A A A G U G A rt G G G A AA AA A G U G A U G G A G A U AC A C C G CC U G S U A G e U A U U G A A A s C U A A G U C A U G U U G A G U U G A U C U U U GA G U G A A A A G G G U A A G A U UC A C UA UC C C n A G A A A A U G U G C U C U e A C UC U U GA U U G AA A A G U U G A A G A A U A U G C U S U U G A A A G C U U G C U G U C U A G G C U A U C U U A G U G A A A A G C G U G A U A A A U U G G e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 3 3 3 73 83 93 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 7 7 7 7 7 7 47 47 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 N 8 8 8 8 8 8 8 8 78 78 78 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000139_0001
' 3 A A A A A A A A A A A A A A A A A A t G AA A A A A A G A G U AC A A C A o U A C A A U G A A U G U U G AA U A C U U U C U G A G U C C U ' A U A C 5 G A U A AC A U G A AA U G G AA U U C U G A G U C UC U A AC A U G G AA U U G A U A A U U U U G A G U C UC e U G A c A U G A U A AC A n G A U G A U A A U A G U G C AA UA G A A G U A UC A U U U U G A G U C A G U U AA UA U C U U U G A G U e U G A U G A U A C A UA G G A U G A G G A A U C U U U G A G u U A U G A U A C A U A U G A UA U U G U A U C U U U G A q U G C U U G A U G A U A C A A U G A A G U A U A U C U U U G eS G C U U G A U G A UA A C AC A U G A U G A A U A U C U U U U G C U U G A U G G U A A A AC A U G A U G A A A U A U C U U d C U U G A U U A U G A U A U C n A U G A U U A A U A A G A A U a A A U G C U U G rt A A A U G C U U G A G A A C U A A A G A G U U A U U C G A A U A S G A A A U G U G A G A C U A AC U G U G G U U A A A es U G A A A G C U U U U G C U U G A U C U G A G A U A AA U A A A U G A U G U G A U G A U A A G C U U G U G A U A A U A A n A A U C G A C A G U G C C e C U G G AA A G U G A A C G A A G U G A S U C U A UA U G UC U U U G A U G U A UA U G A UA U G U G C U C C U G A A A U G C U U G A U A U A A U G A A U G U e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 6 6 6 56 66 76 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 7 7 7 7 7 7 67 67 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 N 8 8 8 8 8 8 8 8 78 78 78 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000140_0001
' 3 A A A A A A A ot A A C A A A A A A A A A A G G U A A A A A A A A A A A A A G ' A AA CA G A G A U G U U G C U U U A U U A U G A C A U U 5 U C A A C G U U C A A C A U C U G U U U A UC U A U U A U G A C AC A A A G C U C U U U G U A C UC U A U U A U G A G A C ec C n C U U C C A A C U U C A A U U U C G A A G G G A G C U U A C U U A U UA G A C A U U UC U U G G U G C UC U A U U A A U G e U C C U U C A A U A U u G U C C U U C G C U A U C U U G U U G C UC U UA U U A U C U U G C G A U U G C U U C U UA U U A q A G U C C U U U U U A U C A U U U U A A U U G C UC U U A U U eS G A G U C C U G A G C U C C A U U A C G CA C A A U U G C C U U A U A U U CC G A C A C U U G U CA C A A U U G C C U U A dn U U G G G A G A G A C U C U U U U U CA C A A U U G C C U U a U U U U U G A G G U G A A U A C G U G U A CC A A U U G C C U rt C U U S U C U U G A C C U G C A U U G A G U A CC A A U U G C CC e A U U C U U G A G C U A C A C C G G A G U A CC A A U U G G s U A U C U U U AA A G C G G U U U G G A G U A CC A A U U U A A U UC U U C G A GA UC AC U C A G G A G U A CC A A U n A UA U A U C U CA G U C G C A A U G A G G A G U A CC A A U e S U G C A A G A A U A U C U A A C A G G U G U G A G G A G U A C C A e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 9 9 9 39 49 59 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 7 7 7 7 7 7 97 97 9 9 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 N 8 8 8 8 8 8 8 8 78 78 88 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000141_0001
' 3 A A A A A A A A A ot G U G AC A A A A C U U C A A A A A U U U U G U G A C AC A A A A A A A A U A U U G U G A C AC GA A AA G U U A U U ' G G U G C C 5 U G G U G C UC U C G G U U U U G U G A C C G A GA U A A G C U U G U G A G G U G U U G U G A C C A G A A U ec C U G G U C A AC U G G U G C UC A U U U U G U U U G U G A C C A G A GA G G U G C AA G U U U U U U G U G A C C A G A A n G A AC U G G G U G G GA G G G U G U U U G U G A C C A G A e U G A AC U U G G U A A G U A G G U U G U U U G U G A C C A C u A U G A G U U U G U G A C C C q A C A U G G G A eS U A U G A C A A A U G U U A U G A C U G A A GA G U U A G G U U G U U U G U G A C U G A G A A U A A G A A A G G U U G U U U G U G U G C d A U U A G C A G A G A G G U U n U G A A A A G U U U U U G U U a U A U U U A U C A A U G A U G A G A A A G G U U G G U U U G U rt U A U U G A U A U G A U G A G A AA AA G G U U U G U U U U S CC U e C U A U U A U GA A A U G A GA G A A G G U U G U U G s G C UC U A U A A A U U A G G A A U G A G AA A G G U U U G U A U G C U U A U U A G G A AA U G G A G AA AA G G G U U G A n U U G CC U U U A U U A G G A AA UA U G A G A AA G G U U G e S A U U G C C U A G C U U A A G A G G A A A U G A G A A A G G U A C e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 1 1 2 12 22 32 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 8 8 8 8 8 8 28 28 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 N 8 8 8 8 8 8 8 8 88 88 88 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000142_0001
' 3 A A A A A A ot A A U A A A A A A A C U A A A A A A A A A A A G U U A A A A C A U A A A C U U A U C AA AA A A U A A U U A U G U G C U '5 GA A U U U A A C G A U U G G CA U C A A G A U A A U U A U G A G U U UA A A G G U C G U G A U A AA UA U A U U A C U C ec A G G U A U A n G A U G A A A A U G U A A U G A U U A U U A A U U UC C C U U U G A G U G A G A UA G A G A U A U U C A U U U e U A U G G U A G U G U C U G G UA G A A A U UC C UC U U u G G G U C U C A A A U U G U A U U U G A U G U A A U UC G U U q U U U U e C U U U U A U U U G A A U A A U G A U A U A C U A C U S U G G U A U C U G U U C U U U C U A A U G A G A U U C U G C G C C C A U A C G U G A A A U d A U G U U C C U A A U G G U A n A G U G C U U U A A G U C A C U G C C U A A U A U U A A C U U ar t A S C A G A G U G C U C C U G C C U AC AA U A G C C U A A G A A A A U AC UC U GC C U C U A A GA C C U A U G G GA A C G es G AC G A C U U U A A C C U U U C U C AC A A G C C U A U A G UC UC A A G C A U C A A G C A AA G C CC A A U A A A U n U G A UC A A U UC U A U AC G C A A G e S G G A C U U A UC A A A A U C UA A U U G C C U C C C A G U A U A UC G C A A GA C U A AA AA U C C U A U U C A U C U G A C A A G C C U C U A A A U e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 4 4 4 94 05 15 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 8 8 8 8 8 8 58 58 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 7 7 7 N 8 8 8 8 8 8 8 8 88 88 88 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000143_0001
' 3 A A A A A A U C U U U A A A A U A A A A A A A C A A A A A A A A A A A A ot U G U U U U A G U A C U A U U U C G AC C A U G A A U G A A ' U U A U U UA U A C U U U U U U G A G AC C A U G A A U G A 5 A U C U U U U A U U G A U U A UA A G A G AC C A U G A A U G ec C U C A U n C U G C G U U CA U C C U U A U U U A G A G C C A U G A A U e U U U C U A A U A A U U U U A A A G A G AC C A A C U G A A G A C U U U U C A U U U U C A A G A G C A AC U G A u U G U U CC A G A A C A C C U U U U C C A A G A G C A AC U G qe U U U U A A U U G A C C A A U U G U G U C C U A G A G C A C A S U A U C A U U U G C G A C U C A A C U C U A A C C A A G A G C A C d G U U A A A A U C A U A U A G A G AA C C A A G A G C A n U G U U U U C U C A A C C C C A A U U G A A C C A A G A G C a U rt U G A G U A G A A A A U A U C A U A U A U U G A U G CC C C A C U A C U U G A C C A A G A G A A C U U G A C CC A A G A S C e G A G A G AA CA G U s A U U A U A C C A C A A U U A C A A CC A A G U U G AC A A AC A U A A A U U A UC U G G A A CC A A U G G A A A A C C G C C C AC U U U U A UC U U G A A C A n U e U A A G U A C U C U U U C C S A C U U G G C A G G A U A A A U U A C U G A A C C U A A C A C C C U C U A U U U U A U C U U G A A e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 7 7 7 77 87 97 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 8 8 8 8 8 8 88 88 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 0 0 N 8 8 8 8 8 8 8 8 88 88 88 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000144_0001
' 3 A A A A AC A A A A A A A A A U U A A A AA A A A A A A A A A A A G A A ot C A G U A A U U G A U A A A U A G A G G A GA A G U G G U C ' A C A U A U A UC G C A A A A C GC C U G G GA A G U U C C 5 A A C A A C U G C A U U U A A A U U A G U U G GA A GA G A U ec G A A C U G A A A G A G U G UA U G A U U A A A A A CA GC G U G GA A U U A U U G A U U U A U U GC A A C G G U G A G C G ne A U G A U C A C U U A C U U U U A A U G A A C A G U G A C U u A A UA A A G C G C U G U U U U UA U A C G A A C G U G C U q G A e U G A A G A G U U U U C U U U A A U U C G A A C G U U CA S A U G U A G G A A A U A G A C U U U A A A U C G A A C G A A d C A U G U A G C U C U G A n A C A U C U A G C U G G G CA U U U U U A UA C G A A C A A G C U U A A U U C A U A G A A U A U a C A C A U U U A G A G AC A G A C U A U U C A A U A G A C C C rt G C A G U A U GA U UC A G C G G A C U A UA U A U GC U A S A G C A es G A G A U A A A U A G AC G A A U A A U A U U A A U U CC A G A A U AC A A U C G U U A G AC A AA U U U A A U UA A C A n A A G A U G C U GA UA U U U A G UC U C U U A U A A U U A e S C A A G U U G AA G C C G U U A U C AA U U C U U U A UA U A U G C C A G U A U A A G U A G U U C U A A C U U U A A U G U e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 0 0 0 50 60 70 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 9 9 9 9 9 9 09 09 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 N 8 8 8 8 8 8 8 8 98 98 98 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000145_0001
' 3 A A A A A A A A U AA AC A A A AA A AA A A A A A AA A A A A A A A A ot U U U C A U U U U A C A A A GA A G A U U CA C U G A C A U ' C U U U C AC UA U U U A C A A A G A G A A U C C U G A C A 5 C C U U U A U C A U U U A C A A A G A G A A G A C CC U G A C ec U C C U U C U U C U U C A U U U A C A A AA G U A C G A G A A C U G A C UC U U C A U U U A C A A A U C A A G A G U A C U G ne G U U C UC U U U C A U U U A C A A A A G A A u U G U U G A U A CC U q U U U C C U U U C A U U U A C A A A A G A G A U A CC e C U G U U G U U C C U U U C A U U U A C A A A A G A G A U A S A C U U G U U C C U U U C U AC U U U A C A A A A G A G A U d A A C U U G U U C CC U U U U AC U U U A CA A A A A G A G A n A A A C U U G U U CC UC U U AC U U U U CA A A A A G A G a U A A A C U U G U U U U C C U U U AC U U C U U A A A A A G A rt C U A A A C U U G U U U C C U A U C A A A U U U A C A A A G S A C U A A A C UC U G U U C G U U C C C A U U U A C AA A A e C s C A C U A A A A C A A A U U U U U U G U U C C U C A U U A C A A C A C U A A CA UC U G U U C UC U U U C A U U U A AC A ne A S A CC A C U G A A C C A C A A A UC U G U U C U U U C U U U U A AC C A U C U A A A U C U U G U U C C U C U U U A C A U U U A e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 3 3 3 33 43 53 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 9 9 9 9 9 9 39 39 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 N 8 8 8 8 8 8 8 8 98 98 98 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000146_0001
' 3 A A A A A A o A A A A A A A t C C G C U A U A AA G A A A A A A A A A A AC A AC AC A A U U U U C A U U A U G U UC U C UC A U U A U ' U C U U C C A U 5 A U A G C A A A U A A A UC U G G U U U C U C U A G U A C A G U A A A G A U A UC U A G U C C AA U A U U ec CA A A U U C A A G U U U G U UA G GC U A U U A U U U U U U U U A G G U n A A G C G U A A U C C U C A U C U U U C G C U A G e G GA A U A G U G G U A A C C CC A AA G C U U U U G U G A u U G A C G G A A A U U U A A C U C U G G U G C U U A G A U G qe C UC U A A G C A C U G G U A U U U U U U G U G C U U A U U U S A C C G G U C U C U U A A C G U U C U A G U G U C A G A A U d U A A U A C A A U U G G U GA C C C C U A G U C G C A C G U A n A U C C G C C A A C U U A G U A A C C A U A G C U U U G A U a G A A A U U U A rt A G G C C A U U G A C U U U C A A U A C G C U A G AC S G A A A C G G C AC A C U U G C CC U U G A A UA A U U A G AC U A U C A U UC A A G U U U G A A U U G G GA G es A G G A A U A A AA A G U AC A G U A U U C U U U G G A U G A A GA n A A C G G A G G G C A U U U U C A CA CC U U U A U U U G A e S A A C C A U U A G U C G A C A G U A A C U A G C A G G U G U C C U G C G U A G C A U G G A C C U C U C C C C U C U G U U U A e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 5 5 6 16 26 36 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 9 9 9 9 9 9 69 69 6 6 6 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 N 8 8 8 8 8 8 8 8 98 98 98 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 8 4 1 8 8 8 8 8 8 8 8 8 8 8 8 8 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000147_0001
' 3 A A A A A o G t A G G A C A U G G A C A C C A C G A A A A A A A A A A A A A A A A A A A A C C AC CA A A A A U C U C A G A A C C A U A ' C 5 G AA U G G A A U G G CA C G C CC G C C C G A A A A A U C U C A G A A C C A U C CA AC A A AA U C U C A G A A C C A ec U U A A U G G A C C C G C A AC AA A A U C U C A G A A C C n U A U A A U G G A C C C G C A C A A A U C A A U C A G A AA C e U C A U A A U G G A C C u U U C A U A q A A U C A C U G G A C C GC C A C G C A AC A A A A U C U C A G A A G AA A UA C U C AC A G e A U A G G A C C U C U S U A A U C G C A C A A A U C U C A U G AA U C A U A A U G G A C C C G C A C A A AA A U C U C d A A G A U n G A C A U A A U G G A C CC C G C A C A A A A U C U A C A G A U C A U A A U G G A A CC C G C A C A A A A U C a A rt G G C A U G C GA A U G A C A U A A U G G G A CC C G C A C A A A A U A U C AC U A A U G G G A CC CC G C A C A A A A S U U U G C A G AA U AC U A AA U U G G A C C G C A C A A A e U s A U U U G C A U U U U G C G A UA U AC U A A U G G A CC C G C A C A A A A A A A U A C U C A A A G G A C C G C AC CA A n U e A U U U G C G A U U C C G C S A U C U A U U U U G A G A U C U A A U G G A C C C A G A A U A C A U A A U G G A C C C C G C G A C e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 8 8 8 98 09 19 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 9 9 9 9 9 9 99 99 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 1 1 1 1 N 8 8 8 8 8 8 8 8 98 98 98 9 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 8 4 1 8 4 1 8 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000148_0001
' 3 A A A A A A A A A A A A A A A A A A A A A A A A U C U C A A G A A A A A ot U C U G U U G A C A U U U U U U A U U C U C A U A G A U U A ' A U C U G U U G A C A U U U U 5 U A U C U G U U G A C A U U U U C U C A U A G G U U U e U A C U U U U U U U C U C A U A G G G c A n C U A U C U G U G A U A U A U C U G U U G A C A U U U U U U C U C U U C U AC U U G A U G C e C C A U A U G U U G A C A U U U U A C A U A U UC U U U G A C A U U U U U U C U C U C A u A C C A A C AC U G A A C G U U G A C A U U U U U U C U A U C q U U U e G A A C C A U A U C U G U U G A C A U U U U U U C U A U S A G A A C C A U A U C U G U U G A C G A A U U U U U U A U A d C A G A A C C A U A U C U G U U C A U G A C U U U U U U A A U n U C A G A A C C A U A U C U G U A U U U U U G A C A U A A A a C U C A G A A C C r A U A U C U G U U t U C U C A G U U G A C A U U G A AA C G A A CA C A U A U C U A C U G U U G A C A U G S A U C U es AA A U C A U C G A A U C A A A C C A U A U C U G U U G A C A U A GA U C GA G A C A A CC AC U A U A U A U C U G U U G A C AC U n A U U G A A U e S C A A A U C U C A A A A U C U G U C A A A A A A U C C A A A A U U C G G A CA CC A U U A U C G U U G C C C U A C A G A A C C A C A U A U U C U G U U U U C U e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 1 1 1 71 81 91 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 0 0 0 0 0 0 20 20 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 N 9 9 9 9 9 9 9 9 09 09 09 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000149_0001
' 3 A A A A A A A A t A G C G A A A A A A A A A A A A A A A A A A A A A A A A o A U U U G A CA AC C U U ' U A U C U G A A AC C U A A U C A A A C A U C U U A A A A UC U U A A U C A A A C A U U U G U 5 U A U U C U G A A C A A U U A A U C A A A C G U U A C A U U A ec A U n C U U U U C U G A A C G A U U A A U C A A G U U U C U G A A A G A U U A A U C A A U G A A U C U U A A U C A A G U C U A e G A U U U U U C U G A AA A G A U U A A A U C A U G U U U u A C A G U U U U C U G C A A A A GA A U U U A A U A A U G C U q G G A e U G U U U U C U C C A A G A U A G A U U A A G A A U U U S U A A G U A U G U U U U C U U C C A A A G A U U A U A A A G C d A U U A A U G U U U U U U C C A A A G A U U C A AA AA U U n U A AC U A A U G U U U U C U U C C A A A G A U U G G A A G a U A G U U A A U G G U A C U U C C A A A G A G U A U C U G A U rt G U A AC UA U A AA U U G CA A C U U C C A A A G C U A A S e U U G G C U U U AA A U A C AC C U U C C A A A U U U C G A s U G A U A A C U U C C A A A G U A U A G C A A G A A C C C C G U A n U U e U AA G A G G A A C UA U C U U A U UC G A A AC CA U U C U U C AC U U U U G C G S A U G C U U A U G A G A U G A A C C U C U U U U C A U U U C U G A A A C A U C U U A A C A U G U C e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 4 4 4 54 64 74 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 0 0 0 0 0 0 40 40 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 N 9 9 9 9 9 9 9 9 09 09 09 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000150_0001
' 3 AA A A A A C A A A A A A A AC A A A A A U U A A U A A A A A A A A A A ot U A C U A CA AC A G A A A A U U A U AA A A A U U U U C U ' G U A AC U U A C A G A A A U U G U U C AC A C A A A U U C 5 U G U A AC A U A C A G A A A U A C G A C AC A A A G U A U C ec U U G U A C A U A C A A G C A U A G UC U A A C A A A A U A U U U U G U A C A G A G A C GA A U U A G A U A A G C U G A U U n C U U U G U A C AA A G A C A A U A A U C A A G A A A G A A eu C C q U U U G U A A A A G A A A U A G G A C C C A U A GA UA e U C C U U U G U A A A A G G A U A U U A A U A AC A U A A U G S U U C C U U U G U G A A A A G G A G A G U A U A A G A A A A d U U U C C U U n A U U U C C U A G A AA A AA G U AA A A U U U A A C A A U C UC A A G G A G AA AA C A G G G G C G C A A a U A U U U rt C C UC A A A A U C U A U U U U C G A A A G G A U G U U G AC A A G C A A A GA A U G G U U GA G C U A C A G A S C CC U A U U U U A G A A A A A A G A C A A A A A A A C A C es U C CC U A U U U GA G A G A A G U C C G U AC G A G U G A AA CA G C U A UA U U U U A G G G A A C U G AC G AC A A A A ne S G G U C C A G G U C U C C C U A C UC U A A G G A AA UA U A U G G A CA C U A C A U C U U G A G G A U C A A U G C A A C A G A e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 7 7 7 37 47 57 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 0 0 0 0 0 0 70 70 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 N 9 9 9 9 9 9 9 9 09 09 09 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000151_0001
' 3 A A A A A A A A A A A A A A U C U A A A U C A A A G A A A A A A A A A A ot U A C AC A U A U U G U C U U U A U G CC C U U C A A A U C ' C U C U G A U U U U C U A U C U U G G CC CC U U C A A A U 5 U C A C U G A U A G U U C U C A U A U U U G C CC U U C A A A e C U U G AC U G A G U G C U C U G A A A U G C CC U U C AC A c C C n U C C U U AC U U A U U C U U A U U A U A U G C CC U U AC e U UC A C AC A U G U G C U C A A U A U A U G C CC U U U u U q A U C A G UC G A A U U C C U A A U A A U A U G C CC UC U e U A U G U G UC U G U U U G C U U G U U A A A U A U G C C C S A U U A A U G A U A G U U G U CC G A A U A A A U A U G C C G A A A A A U C A G A U U U U G A A U U U A U AA A U A U G C dn A G U G G A A U C U U G A A U U U G U A AC U AA A U A U G a U A A A rt A U G C A G A C U S A A A A A A G G C A A C G U U A G G A AC UA U A A U A UA G G U A A G U CA A G A C U U A A A UA U es AC AA U U G A A U U G AC G A A C U U A G U A U C G A G G A C U U A A A A A UA G AC A U G A A A G G G A AC U U A A A n G C A A A AC G U e S A G A A G U A AC G A U A G A A G C U G G G A AC U U U A A G UA C C U G U G AC G UA U G G G A AC U A U U C A C U G U A A A A G U A U A U A A A A U G G G A C A U e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 9 9 0 10 20 30 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 0 0 1 1 1 1 01 01 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 N 9 9 9 9 9 9 9 9 19 19 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000152_0001
' 3 A A A A A A A A A G A U U U AA A A A U A A A A A A A A A A A A A A A A ot U U U A G A U U A C G C C U U A C U A G A C A U U C U U C U ' C U U 5 U U U GC A G GA A G U U A A U A A C A U C C C U A U C U A A A C A U U C U U C U e A U U UC U G A A C A U U C UC U c A A A n A A UA AA CA GC A G GA U U A G A A U A U U C U C A U C U A G A A C A U U C A U CC U A G A A U C A C A A C U e C U U U uq U A A U A A A C G G U U A C U C C U U U U A G A A AC A U A U A A A C A A U G C C U C A U CC U U A G U A G A A C A e U UC U G U A A G G U A A G G U U U A C U C A U CC UC U U A G A A C S C U C G A G C A A A C A A C C C U U C C U U A G A A C A U U U A U G G A G G C G AC A AA U C A U U C C U U A G A dn C A A U U A C a G A A U U U G G A A A A U A A A U C A C U C A U U C C U U A G C U U U A G U U U U C A C C C A U U C C U U A rt U C A A C U G C A U G A A U U U U U A A U C C A U U C C U U S A U C U A C U U A U G U A U U U U U C A C U C C A U U C C U e U s A U U U U A C U A U U G A A U U U U U C C U C C A U U C CC n A C e A C U C C U U U A U U U CA U G U U G C U U U U U AA A C A A U C C C U C AC U U A U U S U G C U C A U U U U U G A U C U U U C A A C C U C U G G G U U C U U U U A C A A A U C U C C A C A e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 2 2 2 92 03 13 2 3 4 5 6 7 8 9 0 1 3 4 6 7 8 9 0 1 2 3 4 5 a 1 1 1 1 1 1 31 31 3 3 3 3 3 3 4 4 4 4 4 4 4 4 5 5 5 5 5 5 N 9 9 9 9 9 9 9 9 19 19 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000153_0001
' 3 A A A A A A A A A A A A A A A ot A C U G A G A U U C AC A A A A A A A A A A A A G U A C U U A U C AC A G A C C A A U U G G G U A C U ' UC A C U G A G A G A U C AC AA G A C C A U U U G G U A C 5 U UC A CA UC G A GA G A U C C A G A C C C U U U U G G U A ec U n C U UC U A U C U C G U A U G A U G A U C AC A GA A C A U C U U U U G G U C G A U G A U C AC A GA G U U C UC U U G G e U UC U U C U A UC A G A U G A U C C A A G U U U U C U U G u U U UC U U C U A G A G A U G A U C C A U G U U U U C U U U q A U U C U U C U U G A G A U G A U C C G U G A G U G U U C U U eS CA A U U C U U C C U G A G A U G A G U C A A G U G U U C UC dn A C A U U C U U A CA UC G A GA A U A UA A A A G U G U U G AA C A U U C U UC U A U G G A UA G G A A A A G U G U U a A G A CA AC U U C C U CA U G A G G UA U C A A A A G U G U rt U A A A A AC U U U U C C U G U A C A A A C C A A A A G U G S U U G G A A A U UC U U C A UC G G G A G A C C A A A A G U e C A A G A CA AC U U C U U C U A UC A G A CA CC A A A A G s n C U e U C U C U U A U GA A AA U U C U UC U C U A UC G U G A G C A A G A C AC A A A A A A S U U C C U G C U U A G A U C A U U U U C C U U U A C A G A C A C U A U C C C A C A A G A C C A C A e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 5 5 5 95 06 16 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 1 1 1 1 1 1 61 61 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 N 9 9 9 9 9 9 9 9 19 19 19 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000154_0001
' 3 A G AC A A A A A A A A AA A A A A A A A A A A A A A A G AA G AC A A ot U G A A U G A A G U U G U G C AC U A U A A G U A A A U G U A '5 U U C U A U A A U U G A U G A GA G AC U A U A U G U G A A C U A G U U A A A C AC U U A G AC U A U A U G A U A G A C A C U A A A G A U U A AC U U U A G AC U A A A U A G A A UA G ec U A U U U A U A U A G U U U A G AC UA U A A A G A U A A U ne G U C u G G G U A C U G G U A A G A G U U G G U U A G C U A U A G U A AA A U U A G AC U U A A U G U U A G A U U G A G U A q U G U G U U A G A U A A C A U G U U A C UA A U U U A A G U eS U U UC U C G U U A G A U U A C A U G U U G C A U G G A A G U U A U C C U G A A U G A G U A C A A U G U A G C A A U G A A d C U A C U A U U G U G A G U C U A A U G U A G U GA G A U G A na U U U U G U C U A A G U U A G U CA A U U U A G A G GA U G rt U U C U G G A C U A AA G A U A G C A U A A G U U U G A G U S G G A A e G U G U A U A G A U GA U A CA U G U A U A GA A G U s G U G U U A U G G U A U G A G G A U U U C A U A G A U A U C U A A n A G G UC G A U G U G U G A U A G A AC U U A U U G A GA e S A G U A C U U U G A U U U U A AA GA G U G A U A U A AC U U U U G A A A U C U U U U C U U A A A G U G A U G U A A A U U U G e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 8 8 8 78 88 98 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 1 1 1 1 1 1 91 91 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 1 1 N 9 9 9 9 9 9 9 9 19 19 19 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A G A A A A A A U A A A A A A A A A A A A A A A G A AA AC A A A A ot U U A U G A U A C U C U U G A U U U C C G G G A A CC C U C ' A UA U U G A U G C A C U U G A U U A U U U G G A A CC C U 5 UC U A U U G A U G U A C U U G A G U U A G C G U G G A A C U e G C U A U U G A U C U A C U U A U C G G U U A G G U G G A A G c n U G C U A U U AA A G C U A G C A C G UC U U G G U G G A UC e A U G C U A U G A U G UC U A UC U A C GC G U G G U G G A u A A U G C U A A A AA U G G C U A UC C A U C G U G G U G A q A A A U G C e U A GA A AA UA U G C U A U U A U C G U G G U U S U A A G U A AA U G A C U G A A G A A A U G C U C U G A U C G U G G U d U A A A U A A G A A A U G C G A U G A U C G U G U n AA GA G U A A U A G U A A G A A A U G U G C U G A U C G U A a G A A G U A A A G A A A G A AA A U U AC C CC U G A U C G U rt G A A G U A CA A U A A A GA G A A A U A A CC U G A U C A S U e G U G A A s G A G U A CA GA U A A A G U A A A G A G A G C U U U A CC UC G A U C U G A C A G U A G U A C A A A G U A C C n G A G U G A A A e S A G A G U G A A U A A G U U A A G U U A UA U U A CC U G U A A CA AC G G U A AA CA U G G A U A C U A A A G A G U G U A U A A A C A G U A A A A U C C A G A U U A C C U e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 1 1 1 51 61 71 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 2 2 2 2 2 2 12 12 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 N 9 9 9 9 9 9 9 9 29 29 29 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000156_0001
' 3 A A A A A A A A A A A A A A A A A A A A A A A t C U G A A G G A A A A A A o U A U A A ' C U A U A G C A C A G C A AC GA C G U U U A C U U C U G A A G G A U A CA U 5 U C U A U U C U G A A G G UA A UA U CA A A A G C A C A G C U U G U U C U G A A G G A U A U e U U C U c G U U C U AC A n U G U C A A G A A C A C A G C G U U C U G A A G G A U A G C A A GA C A C A G C G U U C U G A A G G A U e C U U U A G C A A GA C A C A G C G U U C U G A A G G A u A UC G G U A A G C A A G C AC U A G C G U U C U G A A G G qe A A UC U G A A A G C A AA G A G A U A G C G U U C U G A A G S U A A C U U A A A G C A A A A AA U A GA C G U U C U G A A U U A A C A U A AA A G C A A U AA U G C A U A G U U C U G A dn U U U A A U A U U AA A G C A G U U A A U GA C G U U C U G a A U U U A C U A A C A U A A G C G G G U A A G C U A G U U C U rt U A U U U U UC U A U AA A G G G G G U A A G C U A G U U C S A U A U U U U e C A U A U G U U C U A A A A U G G G G U A A G U A C G U U C U U C U UA A A U U U G G G U A A U GA G GC U s C n C CC A U A U G G U U C U UA U C UC U G G G U A A A G GC e S U C C A U C A U C U C C A C G U U C A G G C U U G G G U A UA U A G C A C A A U C U G U U U C U A C A G U C U U G G G U A A U A e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 4 4 4 34 44 54 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 2 2 2 2 2 2 42 42 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 N 9 9 9 9 9 9 9 9 29 29 29 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000157_0001
' 3 A A A A A A A A A A A A A A A ot U C U U C U AC A A A A G U A A A A A A A A G C C G U A UA U U U U C A A A U C G A U G A U U U U A G C ' U U 5 C U U G U U U U U C U A G G GA U G U G UA UA U G UC A U C C U U UC G U A U U U U U C U A A G G A A G U G G U A U G ec U A C n A U U G U A UA U U U U AC U A U A G U A G U U U U A U U A C U C UC G U U A U U U U C A U A U U A GA G A U U A e U A U A UC U U UC G u A G U A U U C U C A U U U U U A G A U U U A U A UC U U UC U G U A U U C U C A A U q G A U A U A C U U C U G U A U U C U C AC A U U U U G A U A UA U U G U G A eS GA G A U A G A U A C U U C U G U U U U C U UC A C A A U A G U G d A G U A UA A C U U C U G U U U U U U U A C A A U A G U n A GA G A U U A CA UC U C U A U U U U G U A CA A U U A G a G A G G A U A G A A A U A UC U C U A A U U A G U CA U U U A rt UC G A GA G G U A U A A U A U U G U U A G A G U U A U U U S e U UC U G A G G U A C U A UC U G G U UA A G A GA A A U U s U U C U G A A A G A UA U A C U A UC U G U A G GA CA AC A U G U U C U G A GA G G A U A U U U C U A U A G A G U A AC A ne C U U C G A G G U U G C A U A S G G A G A U A C U C C U A G U A C C G U U U C U G A A G G A U A U C U U U C U C U A C A U A G U A e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 6 6 7 17 27 37 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 2 2 2 2 2 2 72 72 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 N 9 9 9 9 9 9 9 9 29 29 29 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000158_0001
' 3 A A A A A A A t A AC A A A A A A A A A A A A A A A A A A A A A U A C U U o C A UC A U G G A A A U A U U G A A U A G C GC U G G U A C U ' U C A UC A U G G A A A U A U U G G A U G G GC U G U A C 5 U U C A U A U G G A A A U G CA UC U G U U U G GC U U G U A ec U U U C C n A U U U A U C C A U G G A A A U A A U U A C U U U G U U G C G U G U C A U G G G A A A U U A C G U U U U G C G U G e C A u U C UA U U C A U A U U U C C U G U G G A G U U A A G G U U U G C G U A UC A U q U U C A U U U C A UC A U G A U G U U G A G A G U U U G C G U A U A A U G U G U G G A G U U U G C e C U U C G U U AC U U A U C U G U U U G S C A U U U C C UC A G G U A A A U G G A G G U U U d U G C C A U A U U U A U G G A U na A U G UC U U AC U U U C AC C U A A U A C U A U G G A G U U rt U A U G UC U U AC U U U A A A A AA U G C U A U G G A G G S U U A A U U U G C AC U U U C A U U U U U G A A AA A G C U A U G G A A e U G C U AC U C U A G G G A C A G C U A U G G G s G A U U A U G UC U U AC A U C U G G G A C A A C G C U A U G G n U G A e U G UA U A U UA G C G UC U C UA U A U U A U G G U G A AC G C U A A G C U UA U S G U U U C U C A U C G A C G C A A G U G A U U A U G U C U U U A U C U A A U C U G A A C A G U C U e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 9 9 9 99 00 10 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 2 2 2 2 3 3 03 03 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 N 9 9 9 9 9 9 9 9 39 39 39 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000159_0001
' 3 AC A U A A A A A A A G U A A G A A AC A A A A A A A A A A A A A A A A ot U C C U U A UA C UC U UC C A U C A C U U A U U A A A U A ' U A U GC U U U A UA G C U AC CC AC C U U A U U A A A U 5 C U C GC G U U A U G G UC C UC U U C AC C U U A U U A A A e A A A UC C G U A UA U G U A C U C A U C C CA U U A U U A A c n U C U A UC U C U A G U A A U G C U C A U C C C U U A U U A U e G C A U A C U C U G U A A U G C U C A U C AC CA U U A U u U U C A q G U C U A C U C U U U A A U G C U C A U C C C A UC U A UA e C C C A U A C U GC G U U A A U U C U C A U C C A UC U U U S G A U C C A U A C U C G U U A A C U C U C A U C C A C U d U U UC U C CC A U A C U C G U U A G C G U C U C A U C C A C n U G A UC U U CC A U A C U C G U U U C UC C U C A U C C A a U U U A UC U CC A U A C U C G U A UA G G UC C U C A U C C rt U G G G U A C U CC A U A C U C G A A U U G UC C U C A U C S A C U e U G U A C C A U A C U C U U AA A U G U C C UC C A U s G G G U G U A UC U C CC AC U A C UC U U U A A U UC U C AC G U C G U G U A UC U U C AC U A A GC G U A G U C A G C U ne U G C U G U A U A U U U U C U S U A U U G G C C G U G U A U U C U C U U A A U G C C C U U C C A C A U C U G C G U U A A U G U C U e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 2 2 2 72 82 92 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 3 3 3 3 3 3 33 33 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 N 9 9 9 9 9 9 9 9 39 39 39 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000160_0001
' 3 A A A A A A A A A A A A A A A A A A A A A A A A G A A C U U A U A A A A ot A CC C A U A U G A A U U U U A A U A A G U U U G A U G U G ' A A CC C A U A U U G A U U U U A A A A A G A U A G A A G U 5 U C A C C A U U AA G G A UA U U U U A A A A A C A G G AA G ec AA G C C A CC CC A C C G C G G G A G G G UA U U U U U U A A U A n C U A G CA AC C G AA A A G G C A C C U AC C A G G G A U U U U A U C U G A A C G e U A G C A C U C U U U U U A C uq U A G A G C A UC C C U AC G A G G A A U U U G U U U G A U A e A U A G A G C U G C C U C GA G G G G G A U U A G A U U A A U S U A U A G A G A U C C C U C A G G G A U AA AA A A U A A A d U C A U A G A U UC G C C C U C A G G G A U A A A n C U C A A A A A A A A U A G A G U G C CC C U C A G G G U U U A UA A A a A A U C A U A C C U C UC G G CC C U C A G G U U U U A U U A rt C U AA U C AC U C U C UC G CC C U C A G U U U G A G A U S C e A U A U AC C UC G U C UC C C U C A A U A A UA C GA A s UA A A A AA UA U AC U G G U C UC G C CC CC U C G A G A C G n C A e U A U A A A A G A A G G U C U G C C U G G GA A GA G A AC S U A UA U A A U U A G G U CC U G C U G CC CC G G A GA G U G A C U U A A A U G A A U A G G U C C U G C A G G A A U U G e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 5 5 5 55 65 75 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 3 3 3 3 3 3 53 53 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 N 9 9 9 9 9 9 9 9 39 39 39 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000161_0001
' 3 A A A A A A A U U A U C U G A A AA A A A A A A A A A A A A A A A A A A ot G U C G G U C U G A AA A U U G G C A U U U A U G A C G C C U ' G G U UA G G U C U G A A 5 U A U C A G G U C A A U U U A C A U U U G A A G U U A G C C A A U U C A C A U U A A C U G C G ec GA G G G U A G G U C n A U A U A U A G G U UC G A A A U G A A U AA CA A C A U G C U G U U A A A C C C A A A C G U A AA G G e G U G C G A U A C G U U G G A U A G U C U G A U G C G A C C A A U C A U A C U U A A A A u G G U C U G U U A A U U A A A q CA U U U A G G A U A G G U C U U U U G C A U U A A U U U A A eS U G C G U A A U U A C G U A G A G G U A A G G U C C U U U G C U A A A U U A G U C U A U A G G U G A U U U G C U A U U U U d A A G G G A G U U U C U A U A G G U C AC U U U G U A A U U U na A A U U U U C G G U A A U A G U A U U AC U U A U U A U U U rt A G G UC G U U U C U GA G A U A U U U AC U U C C A G U U S U G G U G G U U U C U GA G A UA A UC e U AC G G U U A G U A U s G G G G G G G U U U C U GA G G A U U A G C U U U A A A C C A G n A A U U U G G G U U U C U GA G U G C AA U AC U U C AC e S C A G A G U G G G U U U C A G U U U U G C G GA A A AC U U A G A G A G U G G G U U U U C U U C A U U G U C A A G A C U A C A e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 8 8 8 38 48 58 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 3 3 3 3 3 3 83 83 8 8 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 N 9 9 9 9 9 9 9 9 39 39 39 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A A A A A A A A A A A A ot A U G U G U U U U U A A A A A A A A A A A A A A A U U A AC U U U U A A G C A A U G U G A A G G G U U A ' U A U G U A U 5 CC U A U G A U U U U U A G GA U A U G U G A A G U G U U A G G A U U U U U A CC U A U G U G A A U U G U ec G CC UC U U A U n U G C C U G A U U U A G A G CC U A U G U G A C U U G U G U A U G A U U A A A G CC UC A U G U G U C U U e G U G C C G U A A G U G A U U A A G A G C U A U G U U U C U u A G U G C G A A G U G A U A A A G A G CC UC A U G G U U C q A A G U G U G A A G U G A U A A A G A G C UC A U UA U G U U eS AA A A G U G U G A A G U G U U AA A A G A G C C U U U G U U A A A A A G U G U G A A G U U U A A A A A G A G C C C U U G d A n A A A a U U A A U G U G A A G U A A U A U G U G A A U U U A A A A A A G A G C G C U U A U U A A U U A A A A G A G U G C U rt U U U U A CC U A U G U G G G U U U U A A AA A G A U U G C S U U U U U C e G C UC A U G U U A A G U U U U A A A G C U U G U U s G U U U G C UC A U G G U U U U U U A AA AA A UA C U U U C n A U C G U U U U U G C U A U U GA U A U U U U U A A A A A UC G U U GA G U G CC U A A A GA G A U U U U U A AA U A U e S A C U A U U C A G U A A G U G C C U G A U G A U U U U U A G U A A e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 0 0 1 11 21 31 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 4 4 4 4 4 4 14 14 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 N 9 9 9 9 9 9 9 9 49 49 49 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000163_0001
' 3 A A A A A A o U A A t C A A A A A U U U C A A A A A A A A A A A A A AC U A U A U U A A U A A U U A A U G A U A G U U U U G G C U U A U G U ' A C U A UA U A A A U G A U U G U U U UA U G C U U A U A 5 U A C U A UA U G A A A U G A C U G U U U U A U G C U U C A ec U U A C G U U A UC A A C U A A U G A A U G C CC U G C G A U A U G C U UC A A AA A G A A U U U U C C U A A U A U G C A G ne U G U U A C U G A A A G A A U U U U C C A A A U A U G C A A u U U G U U A C A C C q C U U G U U A A A AC A G A A U U U C U A A A U A U U G eS U C U U G U U U U U C U U G U U C A A C A A A A C A G A AC A U A U U U G U A A A U A U U A A U A U U G G U A G AA AA U U C d G U U C U U G n U G U A A A C G A U A U G G U A U A U G G G A U A A CA U a U G U U C U A U A A A U G G G G U A C G U U UC A U A A U A A A U G A U U U U G A G G U C A rt UC U U U G U C U GC A A A U A A A U G A G U U U G G G U C S G U U e C U U G U U A A C A A A U G A A U G U G G U U G G U U s U G C U U G U C G U UC C A A A A G A AA U A UA U G U U G G U U U G C U U G U G C UC C AC AA A A G AA U A U G U U G U ne C U G C U C U U C A A G U U C S U U G C C A C A G C U A U G U U A C U U G U C U U U U G C C U C U A A A C A A A U C U U A U G U A e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 3 3 3 93 04 14 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 4 4 4 4 4 4 44 44 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 N 9 9 9 9 9 9 9 9 49 49 49 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000164_0001
' 3 A A A A A A A A o C A A A A A A A A t U A U C U U A U A U U U C A A U A A A A A A A A A A A A U A U U U C A A A U U A UA A G G A U A U ' G U C U C U U A U 5 U U A G U C U U A U A U U U C A G A U U U U A U A A U A U UC U A U A U U U C G G A U U U U A C A G G A e A G c U U G A U G U C C U U A U A U U U AC G G A G A U U A A U U U n A U A G G U U UC U U A U A U U A AC G G G A A A A A U A e G u C A C U U A U A GA G U C U U A U A U A A AC A G G U G A G C U G G A U U A G U C U U A U A U A A C A G A A AC G U A qe U GC CA U G GA G U A G U C U U A U U U A A C A U U A A U U U S A C A C U G A G U A G U C U U A C C U U A A C U A U A C G d A U U A C U G A G U A G U U U n A A U A A C U G A G U A G U C U UC C U U A A A U A U A G U A G U C A U C U U A G U C A G U a G AA U U A A C U G A G U A G U U CA U C UC U G A A A AC C rt AA G CC U A A C U G A G U U A G C U U U C U C A U C A G U S GA U U CC U A A C U G A G U A G U A A C U CA U UC CA U e G A U C U CC U A A C U G A U U U A C A G U C U A C A A GA C s UC G G A C CC UC A A A U G G A G C UC U U U A U G G U A G ne U G A U C U A C U G A U U U G S U C U C C G U G A U C U C C U A C C U A A U U C U U G C U C U G U U C C U C U U U C A A C A A A C e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 6 6 6 76 86 96 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 4 4 4 4 4 4 74 74 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 9 N 9 9 9 9 9 9 9 9 49 49 49 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000165_0001
' 3 A A A A A A A A A A A A A A A A G A A A A U U U U U C A A A A A A A A A ot A U G U U A U U U A U G U U U UC U U A CA UC C CC U U U A ' A A U G U U A A G U U U G U U U UC U U U A UC U A U C A 5 AC A A U G U U U A A U U G U U U U G G U U C U U A C U U U U ec U AC AA A U G U U U U A G U C A U U GA U U G U U UC U U U A A U U A A A U G G U A U A U C GA G U G U U C U U U G A U A n U G U C AA A U A U G U A U A U C A G U G U U C U U A U C A eu C U G U C A AA A U U U A U A U C A G U G U U C U CA A A A q G C U G U C A A C e A G U U A U A U C A G U G U U C U G G A U U S UA G C U G U C AC A U G U U GA U A U C A G U G U U U C A C d U G C U U A C G U C A U G U G G U A n U A U A U C A G U G U C A C U A G G G U G U A A U G A GA U A U C A G C G U C U G U a UC U U C U A C U G C A A U C GA G G U A U AC G U UC U A U rt A U U A UA G C U AC A A U C A A G U A UA U AC G A C C C S C e U A U U U G G UC G AC AA U U C GA A G U A AC U C G A s A C U A U U A U U C A U G C G U U C GA A G U U U A U U UC U C U A U A UC U A U U C G U U U G U U C G A GA G U A A A A U ne A A A U U U A G UC G A G U A S U U C U U G A U G U U C A G A G U C U A C U A U A U A C U U U G C U U A U G U U C A G A G U U U G e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 9 9 9 59 69 79 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 4 4 4 4 4 4 94 94 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 N 9 9 9 9 9 9 9 9 59 59 59 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000166_0001
' 3 A A A A A A A C A A A A A A A U A U G AC A A AC U A A A A A A A A A A ot U UA U UC G U U G G U A U G UC U U U C U U C U U A A U U A A ' C G U UC G U U 5 G U G U A U G UC U U U A U U C U U A A U G U G U U U UC G U U G U A U G UC U U A A U U C U U A A U ec U A U n G C C A U U UC G U U G U U G U A U G C U G A A U U C U U A G A G A U U C U G U U G U A U G C A GA A A U U C U U U A e U U A A G A U U C U G U U G U A U G UC GA A AA A U C U A u U U G C A G A U U C U G U U G U A U U UC G U U C A G A A U q U U U A C A G A U U C U G U U G U A A U UC U U U A G A A U U eS A A A U A U U G U CA A G A U U C U G U U G U C A U C U A G A A G G C G G C U A A G A U U C U G U U G A C A U C U A G A A d G C n U A U G G U A AC G A U U C U G U U C A C A U C U A G G a A C AC A U U G G U A C G A U U C U G U U C G A C A U C U A A rt C U U A A U U G G U A C GA A U U C U G U C C A C A U C U G S A G U A A e U U G G U A C G A U U UC U G U C AC CA A U C U A s A U U U A A U U G G U A AC G A U U A U C U G U U C C A U UC n G U A G U A e A G U U G U AA U U G G U A AC G A U A A U G C U G U AC C A A S A U U G G U A AC G A G G AC U G G U AC C G G U G U U G U A A U U G G U A A G C A G U G G A U C A U G U A C U e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 2 2 2 32 42 52 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 5 5 5 5 5 5 25 25 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 N 9 9 9 9 9 9 9 9 59 59 59 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000167_0001
' 3 A A A A A A U A A A A A A U G G A A C A A A A A A A A A AA A A A A A A ot A U U A A U U A U A U C A A C A U U U C C U C A U U A C U G ' C UC A U A A A U U A U U C U A U U U C C C A C UC U G C U 5 A U C A A A A A UA G U U A U C U A U U U C G C A A UC U G U ec A n A A C G A A A C U A U G A A A A A A A A UA C C C U A U U U U A AC A A U U C U A A U C C U U U U A e G U U A U G A A A G A G U C U C C U U U A C A U G A A U u U U U C C U G A A A A A C A C C A U U U A A A C A A U q G A UC U U A C A U U A A A A A A e A U A C U A U A C U UC G A U U G U G A U AA UC U A C C UC U AA UA U A A G U A U A A S A U A G A A C U A U C C U U A d U C U U U A G U U C U G C U A A A C U C U C G CC A G U C U A n U G U U A A A UA C G C U C A A A C A C U G G G A G A A C U ar t G U G U A U U A C A U A U G U A U U A A A A A A U A C UC U U G G A A U U A G C A G U G U A C A A A C U A C U U U U C A C S G C A U U G C A G U A C A A C A A A C U U U U U U AA CA A es A A G C C U U U G C A A C A A A G G U G U U G C U G U C A A C A A A C U G G U U A A A A A A AA CA A A AA U U A GA U A C n U A U U G G U U G U A CC A AC A A A AC A A U A A A GA U A e S A A A G A U U U A U G G U U A U A A A C A A A C A A G C A A A G A e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 4 4 5 15 25 35 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 5 5 5 5 5 5 55 55 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 N 9 9 9 9 9 9 9 9 59 59 59 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000168_0001
' 3 A A A U A A A A A A A A A A A U C A A A A A A A A A A A A A A A A A A A ot A A A U A U U C A U C A U C U U U A C A A G C U A C U U A U ' U A C A U A A U C A U C C U C U U U A C A A G C U A C U A U 5 A A U A A U A A U C U A C U U C UC U U A C A A G C U A C G G ec A U U C A A U A A U C A U U C UC U U A C A A G C U A A U U A n A C C U C A A U A A U C A U U C AC U U A C A A G C UC U A e G G U U U C A A U A A U C A U U A u U G G C U U C A A U A A U C q U U U U C U U C A A A U U C U U A CA AC AA G C A A U AC U U A C AA GA A G e U A A U C A C A U AC U U A U U A C A U A S A U G A G U C U U C U G U G U C U U AC A U A A U C A C A U C A U U A C C U d U A U C U U AC A U A A U C A C A U C A U U A G U n U A U G G G U C U U AC A U A A A C A C A U C A U U A C a U U U A U A U G A U G U C U U C A U A A A C A C A U C A U A A rt UC G G A U G A U G U C U U C A U A A A C A C A U C A CA U S U UA AC U A U G A U G U C U U C A CA A A A C A C A U C C es G A G GA U A G G C G U G A U G U C U U AC C CA A A A C A C A U U G A U U U G A U G U C U U U C CA A A A CA A C A UA A ne S U A A G U G C G G U U G A U G U C U U C C AC A A CA AC C C AC C G G A C A G U U G A U G U U C A U U A C A A C A A A A C U A e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 7 7 7 97 08 18 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 5 5 5 5 5 5 85 85 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 0 0 0 0 N 9 9 9 9 9 9 9 9 59 59 59 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000169_0001
' 3 A A A A A A A A A A A A A A A A A G C C U U G C A A A A A A A A A A A A ot A U U A A U A G C C U U U U U G C U U U A A A A U C C A U U ' U G U A A U U A A U A G C C U U G C U U A A U U A U C A A U 5 U A U A A C C U A G C C U U G C U A U A A U A U A U A G A U U e U G G U A A A U C U A G C C U U G U U G U UA C C A U A A A C c n U U UC AC AA A A U C U A G C C U U C A A UC C C A U U A AC e A U U A C A C A U C U A G C C U G G U UA A G C C A U C C u A G U A A A U C A U C U A G C C U AC U U A U G C G C A U C q A U U U A C U U C A U C U A G C U U A U G CC U C C U G G U U eS G A A A A A U U U U U C U A A U U U U U AC U C U A G C C G A A C C C U U A A U U U AC UA C U A C U A G C U C C C C C A dn U A C A A U U U U U U U C UA C U G C G A G U U C C A C A a U G A U A U U U G A rt C A U C U U U U U U C UA C S A U C U U A A A G A G U U C U U U A U U U U U U U U C A U U C A UC U G U U U A e U A U C U C UC U U U U U U U C C G U C A C U G U U G U G s C U C C A U U C U G U C U U U U U U U UA A C U U A C U G U A U n G A A G C G U U U C U U U U U U C U UC C U C AC C U C A A e S A A U A C G U G G UC C U U A U U U C U U U U U U A UC G G A G C C A U G C U U U U C U U U U U C C U A A A C A G A C A C G U e 1 .4 1 .5 1 .6 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 0 0 0 70 80 90 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 6 6 6 6 6 6 16 16 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 N 9 9 9 9 9 9 9 9 69 69 69 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000170_0001
' 3 A A A A A A A A A C U A A A A A A AA A A A A A A A A A U A A A A A A A ot U UC U A C U A A A A U A A U A G G U G A G A U A U G A A A U A G UA A A U U U U A A U U ' U 5 U U C U A C U A A A A U A G G G U U C U A C U A A A A C G A G U G A A G G G A A A AA UA ec UC C U A U A G A G U A G U G G G U U U C n A U U C U A U A A A C C A G A G A A U A U G U A G A G U C U A A G U A A A U A U A G e C G G U U U C U A C U A AC U C A u C G G G U U U C U A C U C C G A A G A U A A U A U A q C U A UC U G G G U U U C U A C C C A U A G G U G U U A G U A U C C A U C U U A A U U A G U A eS U A U U G G G U U U C U A U U U G G G U U U C U A U C A C U A G U A A U G A G U G G G U C U C U C A C A A G A A A A G A G dn A C U U U U U U G a A G G U U U U A C U C A C A U G U A A G A G A A U C U U U U G G G U U U U A CA U C A U U A A A U G A G rt U U C UC U U U G G G U A U U U CA U G C A G G U A G U G A S UA U U U U C U U U G G G A U U G e U U A A A A A G U U G U G s G U U U U U C U U U G G U AA UA U U U G G G UC A C U A U G U U C U U U U U C U U U G A A A U U A A G C A A G C U A A U G ne A A C U S C U U U C U U AA UA U A UA U G A U G C G A CA UC G C C A C A U C U U U U U C U U A A A U A A A U C G G U G G G A A e 1 .2 1 .3 1 .4 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 3 3 3 53 63 73 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 a 6 6 6 6 6 6 36 36 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 N 9 9 9 9 9 9 9 9 69 69 69 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000171_0001
' 3 A A A A A A A o C U U A A t G U G AC AA AC U A A C U A A AC A A A A A A A A A A A U C U U A G U G C U U U U A U U CC A A A C U U C U A C AA '5 U U C U U A G U G C C U U U G A G U CC A A A C U U C U A C U U U C U U A G U U C U C U G U C A A A C U U C U A ec A U U U C U U G A A U U U U U A G U C C U C U U A U A A U U G C C U G U C A A A C U U C U U U C C U G U CC AC A A C U U C ne G A A U U U C U U C A C U U CC A C C U G U C AC A A C U U u A G A A U U U C U U U U C G U A A C C U G U C AC A A A C U q U A G A A eS A U A G A U U U C U C U U U A A U U U G U A U U U A A C C U G U C C AA A A CA C A A A U U A U AA C C U G U C C A A dn G U U A A G A A U U U A G C A A U U A U U A C C U G U C C A a A G UA A G A A U C U G U A U GC U A U U U A C C U G U C C A U U A G A A A U A U UA A A U GC A G U U U C C U G U C rt G G GA U A U A G A G U U C A U A G U C G U A C A A C U G U S A e G A s U G G GA U A UA A GA GA A G A C U U C U G U C A A C U G A G G U G A A U U U A U A G C G U U G U U G U C U U U U A CA C U G G U A G U A U G U U U C G U G U GC U U U U A C C n G G U G A G A U G G G U AC AC A U U U G U GC U U U U AA C e S U G G G A C U G U G A G A G U U U U G U U A G U U G U C G U U U A e 1 .0 1 .1 1 .2 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 6 6 6 36 46 56 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a 6 6 6 6 6 6 66 66 6 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 N 9 9 9 9 9 9 9 9 69 69 69 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000172_0001
' 3 A A A A A A ot AC A A A A A A A A A A A A A A A A A A A A A A A U G A A C U U C U U A U U C G U A U U C A CA A A G A A U U U U C U U ' A C A A U G A A C U U U 5 C A C A A U G A A C U U C U A A CA AC A G G A C C G U A U U UC U U A A AC A A A G U U G C U A ec A C A n U A C CA A A U G A A C UC U U UC U A A C A A U C A C G U e C U A C C C A A A U G A A U U A C C G A A C U C U A A C A G U G A A G A A A U G A A C U U UC U U A A C A U A C G A u U U C U A C C AC A U A A G A A UC U U C U U A A A G A U A G q UC U U C U A AC A C A UA U G A A C U U C U U A U A U U U A eS A C U U C U A C A C A A U G A A C U U C U U U A A U U G A U C U U C A C A A U C U U U U A d A A U C A A U G A C U n A A A C U U C U A C G G C U a C A AC C A A U G A A C U U C U U U U A A C rt C AC A A S U C AC A C U U C U A AC CA A A U G A A C UC A U C G C A A AA C U U C UC A C C A A U G A A A UC G U U A G C U C C A A C U U UC U A AC C A A U G A A A A A C A G G es G U G U C C A A C U U C U C U A A C C A A A U G G A AC G U U U G n C G U C AC A A e S C U A A UC U C U A C C A A U U G A A U A U U A C C U G U C A U C U G U C A A C U C A AC C AC A A U A AC G U U U C A C A A A U C U U U C U A A C A A C A A U A A G A U e 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 8 8 9 19 29 39 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 a 6 6 6 6 6 6 96 96 9 9 9 9 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 N 9 9 9 9 9 9 9 9 69 69 69 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000173_0001
' 3 A A A A A A A A A o G U U AA A A A A A A t U G A U G G C A G A U A A U A A A A A A A A A A A A U U A A U G G C A G U U A A C U AA G U U A A A A C C ' A A U G G C A U U U U U U U A U U U A A A C C 5 U U G U U U U U G U U A A U G G C U U U G G C G U U U U U U A A U G ec A U UA U U G U U A A U G G C U U U U U U U A U U U U A U U U U U G U U A A U G U C U U A A A U C UC A U U U U AA C n G U A U U U G U U A A U U U C U U G U G C C UC A U U U A A eu A G U A q U U U G U U A A U U U A A U U G C C U A C U U A A G e G A G U A U U U G U U A U U U U A C U U U C C UC A U U A U A S A G A G U A U U U G U U U U U U U U A A U A A U C C C U U U d U A G A G U A UA U G U U G A U U G U U U U AC A U C C C U A n A U A G A G U U UA U U U G A U U A C C U A AC A U C C U A a UC A U A G A G G U A U U U G A G C U U A A A A AC A U C A U rt A UC A U A G A A G U A U A U G A G UC C U A A AC A U U A S C e A U A U A G G A G U A G A UA A C U U C G U A A AC A C A s G C C G G A U A U A A C C G A A G U G G U A A A G G G G U A A AC CC C A U A UA U A G A G GA A A CA A U A A G G U A A A ne U G G C C U A A A G G U A U A S C U G C G G C A A U A G G U A A U U G G A C A U C U A U A G G A C A A G A A U C U U A G G U A U C e 1 .6 1 .7 1 .8 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 1 1 1 91 02 12 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 a 7 7 7 7 7 7 27 27 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 N 9 9 9 9 9 9 9 9 79 79 79 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000174_0001
' 3 A A A A A A A A A A A A A A A A A A A U AA A A U U C A t C A AA AC A A o A C U C U U U C A G U U G ' C G C U C U U U C A G U U G U U A U U U U C U G G UA U AC U 5 C A U C U C U U U C A C U A U U U CC UC U G A U AC C G U U G U A AC U A U U U C C U G A U ec G U CC U U C U U U U C C U U U U A G U C A G U U A G U A U U U C C U G A n C U U C U U C U U U C A G A U U G U A U U U C C U G e A G U U U C U UC U C U U U C A A A C U G U A U U U C C U u A UC C U U U U U U C U C U U U C G A A C U G U A U U U C C q G A A C U U A C U U U U C U C U U U U G G A C U G U A U U U C eS A A A U A C U U U U C U C U U A G U U G A C U G U A UA U U d U G C A U A C U U U U C U C A G U G A C U A C U G U U U C U A n A A G C A U A C U U U U UC C U G U G A A U G A A UC G G U a A U A G C A U A C U U U U rt U A C A G C A U A C U U U U UC G U G U U G A G G A U U A C U S A A C C U U U G A G UA G U G U U G A C U e A s C U G C A G C A C A U A CA U U U U U C A G U G G G U G A C U G C C G C AC U C U U A U C U C A A U U G U G A C n A A U e A A S C C G C AC G C A A A UC U GA U U U C GA A A U G U G A A UC U A A A U G C A G G C U U A UC C G U U U C G G A U G U G C U G C C A C A G A C A U A U A C U U U U A C A G A U G U e 1 .4 1 .6 1 .7 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 4 4 4 84 94 05 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 a 7 7 7 7 7 7 57 57 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 7 7 N 9 9 9 9 9 9 9 9 79 79 79 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000175_0001
' 3 A A A A A A A U G G U C A A A A A A A A A A A A A A A A U A U A A A A A ot' U U G G U A U G C A A U U G G C U C C C U U A A A U U A U U 5 AC U U G G C e A U U G C C U G C A A U U G G C U C C C A U G C A A A A A U A CA U G C A A U U G G C U C C G U A G G A A A c U n A C A U U U C A U A C U C A C C A U G C A A U U G G U A G G C G U U U A G A A C U G C e G A U C A A U C C A C A A U U G G G U C U A G A A UC G C AC A U U G U G C A U G A G A U G A u U G A U C U A U C q C C A A UC G G AC AA U U U A G G A A A U G e U G A U U A A U C C A A U A U C U G C A A U U U G A A A A U S C U CC U G A C U G C A A A U C C A A C U G C A A U U U G A A A A dn U U C C C A U C A U A A A U C C A A C U G C A C A U A G A A A U U U C C A C A A A U C C A A C U G C U U A C A G A A a U A A U U C U G C A A A A A U C C A A C U G AC U U U C A G A rt U UA U U U U G G UC A AA A U C C A A C U C U U U C AC G S A U U A G G G UC U A AA A U C C A A C GA U C C U U A U C e G U s U U A U G C G G G C U A A AA U C CC A A A AC U U C U U n C G e A U G U A A G C G U G U G GC G G C U AA A AA U C A U C AC G A G C AC U C U A U C U S C U G G G C U A A A A U C A A G A C A U C G A C U G G G U G C G G G C U A A A A U G A A U A C A U e 1 .3 1 .4 1 .5 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 7 7 7 67 77 87 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 a 7 7 7 7 7 7 77 87 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 0 N 9 9 9 9 9 9 9 9 79 79 79 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000176_0001
' 3 A A A A A A A A A A A A A A A A A A t U U U A C C C AC A A A A A A A A A o U A G A A G G A U G U ' U U A G A A U G A U U U U U U A C C AC U C U A U G C AC 5 C U U A G A U U G A G U U G U U A A A C C U U C U A U G G U G A U U G U U C A A C AC U U C U A UA U ec AA CA U U A G A U C U C U U A A A A U U C U A U U G A U U G U A n G A U U U C A U C A U U C U A A C A A A U U G A U U C A U C U e A A U U C G A A A U U G A U G U U U C U U U C UA U U C u A G U A G A A C A G A A A U U G A U U U U U U U U U C A U U q A AA A G A A G A G A A A U U G U U U U U U U G U U U U C A U eS C A A A G A A G A G A A A U U A A A U U U A U A G U U U U C A d A C A A A G A A G A G A A A U G U A U A U C A G U U U U C U n A A CA AC A A G A A G A A G A A A U U U A A A C A G U U U a G A A A AC A A G A A GA A GA A A U U U U A U A C A G U U U rt U G G A A AC GA A GA G A G G A A G U U U A U A C A G U U S U U e U G A A G A G AA GA A GA A G G U U CC A U A C A G U A U s G A U U G A G A G A G A G A G A A U G G U U CC AC U A C AC G n A A U U G UC G G A G A G A GA GA U U G G C A U A A AC e S C G A A G A U U C U G A G A G A G U C G G U U G C U C A U U A C A G A U U C C U C U G A G A G A U G U U U G C G U C C A C A U e 1 .1 1 .2 1 .3 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 0 0 0 40 50 60 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 a 8 8 8 8 8 8 08 08 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 N 9 9 9 9 9 9 9 9 89 89 89 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A A A A A G G U A A A A A A A G AC A A A A A AC A A A A A A U A ot U G U AC U G C G A A G C AC AA UA G A A U A G U C U U A ' AC UA U U A A U G A U U A C GC G C A U G C U A G A U G U 5 G C A U A A AA U AC A A U U C C G C A U G A C U A AA C G U ec U G C A U AC A A G A A C U U C C G AC A UA G A C U C AA A U n A U G A C A C AA UC U U U U U C C G AC A U G A C U A C U e U A U A U A A C UA A A U u C U U U U C C G C A A U G A U C U U U A U G U A A A U A q U C U C U C U A A U A AA C U U U U C C G C AA U A G A U U G U A U C U U U U C C G C A UA U U U G eS U U C C A U U G U U C U C U C U U U C U U U U C C G C A U A G AC d C n A U U U G U C A G U U G U A A G U G U U C U U U U C CC G C A U U U a U C A U C G A A G U U C U UC U U U C U U C G G U G U rt U U C U A U U G C A A U U A G U U U U U U U CC A A U U S U U U G A U U U U G U A C U A G C U U A G C A G C U U U A G e U U U U A U U U G U s G U U A A A U C U U U U G U C A U C U U U A A U U UC A UA U U A CC A U U UC U GA U U U C A U U G G G n A G A A U U A A U U C G C G U C e G G A AA A U GA U CC AA A U U U A G U U U G A U U S C A A G A A A U U C A G U U C G C C A G U U C U A U U U C G U A A A U U U C U A G U U U C G U C U e 1 .9 1 .0 1 .1 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 2 3 3 23 33 43 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 a 8 8 8 8 8 8 38 38 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 N 9 9 9 9 9 9 9 9 89 89 89 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A A A A U A A AC A A A A A A A A A A A A A AC A A A A A A G G A ot A U U C ' A A U U U A A A U C A A C U A A A U A A U C A A A A U A G A A U A G C U U A G U G U A U U A U AC A A A A UA G 5 C A U A UC U U C CA AA A A U G U G U A U AC A A A G UA G ec C G U n U U C A A A U C A C U A A G A U U U A G A G U A U AC A A A G UA U U A A A C G A G A U U C UC U G U A U AC A U A G e G U U A U G C C U A A A C A A G A U U G A G A U U U U C U U AA CA UC G U A U AC G U A u U A q A G A U A U A C U G U A U U G U e U U U U U G U U C UC U A C U U U A A A C U G U A A U G S A A G A G C U U C U U A G G A CA A C U A A A C U G U U A U d U U AC A U C n U U U A C A C C C U U U G A G G A A U A A A C U G A U A A U U G C A C C G A A U U AC A U A A A A C U A A U a U U U AC G UC A U G A A A U A A C A A G C A U A A A C A A A rt U U U U U U C A U G A UC C U A G G A G G C A U A A A A A A S A A e U A C C A C A U G A A G U A U A C G G G C A U A A AC A A s G G A U GC A G A C UC U A UC U G U U G G G C AA U A U AC A n G G G U e U CA G G A U A A A C U A A A G C G U A C U U G G A U U G G G C AA UA A U AC S A A A A U A A U U G G G C U A U C A U G U G U A C A U U C U G G C G A U C A A U U G G G A C G U A e 1 .7 1 .8 1 .9 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 5 5 5 06 16 26 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 a 8 8 8 8 8 8 68 68 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 N 9 9 9 9 9 9 9 9 89 89 89 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 A A A A G A A A G A A A C A A A U U U A A A A A A A A A G A A A A A A A ot AA U C A U G AC A U U U U U U A U U U A U U C U A U U U U ' A CA U U A U G C UA U U A G U A U U U UA U U C U A U U U 5 C A CA C U A U U G C A U U A A G U A U U A A C UC C U A U U e U A A U A U A U G C A U A A A G U A U A U C A U U A A C U A U c U C A U A A U A U G C A AC A A A G U U C U A G G A A C U U A ne U U C U A A A U A U G C C A A A A G C U u A U U A A A A A U A U G A U A A A A G A U A G A A C C q A U U A G A A A A U A A A A A A U A G A A UC U e U U AC U A A U A A A U A A U A G A A C S G A A A U U U G A A A U G U G A A A A A U A A U C A U A A G A U A U A G A A d G A G A A A U U G U C A U A U A A U A U A G A n G A A C UC G U U G A A A U A G U C A U A G C AC U A U A G a AA G GA U U UC G G AA A U A A G U C A G U G G AC U A U A rt G A G U U G U U G AA CC C A A G U C AC C AA A G A U A U S e U A A U GA U C UC G G U G G G C A A G U U C U G A A G A U A s G G A G U GA U U UC U G U A A G C A A G U U U G A A C G A U n G U G U C U GA G U C U G U C A G C AC AA C U U G A A C AC e S A G U A U C A G G G U U U A G U C A A CA AC G G C U U G G U G A GA G C U A G U U C G A C A A A C A G G A C U C U G U G A A e 1 .5 1 .6 1 .7 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 8 8 8 88 98 09 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 a 8 8 8 8 8 8 98 98 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 1 1 1 N 9 9 9 9 9 9 9 9 89 89 89 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 x e 1 l 4 14 14 14 14 14 14 14 14 14 1 9 4 1 9 4 1 9 4 1 9 4 1 9 9 9 9 9 9 9 9 9 9 9 9 9 4 14 14 14 1 1 1 1 1 1 1 1 1 1 pu 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 D - D - D - - - D A A A A A A A A A A A A A A A A A A A A A A A A A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-10312
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' 3 ot au a c a au au au aa a a a a a a a a a a a a a a a a a au a a u a aa u g a c ' u u c c c u u u ca a c a g c u c a c u c u a u u u u aa a u a g 5 a c u u c c u u u a a c a ga g u c a a u c g g u g u a a a u c a ec u a c u u c c c u u u a a c a a g u c u a a u u a g a a ca u u c n a e a u a u a c u u a u a a c u u c u a c u c a u a c u u u a u a c a c u a u a a c a c u u a a g c u c a g u c u u a g a a c aa ga c a a a u u g a u u a u c g u a c a a u a a a ca a a c u a u a c uq a f u eS A f a f a f u f c f a u a u f a c u a a A f u u u a f c f u f u f f U f A f f c A f f U f C f a A f u C f c f a f u C f u f f f a U U A f f f f f f f C f C f U G f A f A f A f U f U f U f U f d C f A f f f A f U f A f A f U f A C f f U A f f A f f f f C f A f U f C f A f U f U f A f G f f U f A f A f U f A f A f U f U f U f n U rt u C A A f f f a f U a A A f u f a f A a A A C A A U U U a u c f C af Ac C A c U C u Ac f U U a f C a U u g f a f u a f u f u f u f S C e u U c U s g u Cu A c A a Ua A c A f a A a f a C f a f a A c C f a A c U f C u f C f f u A c a U c Ca Au U f Cu U c U U a U c u n u u c u e a g u g ua g c u c a a u u u g c u u c c c c u u a c a c a u a c a a a c a c a a a c a c c a a a c a c u u ac a c u g a a u c u c a ca u c u c a u c u c c gg u u u c g a a g a a g g u g u u c u S u g a u u g u u c u a u u a c a a c a a a a a a c a u c a u c a u c e 1 .5 1 .6 1 .7 1 .8 1 .9 1 .0 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 8 8 8 8 8 9 19 29 39 49 59 69 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a 5 5 5 5 5 5 5 5 5 5 5 5 95 95 95 06 06 06 0 0 0 0 0 0 0 1 1 N 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 69 69 69 69 69 69 6 6 6 x e 1 l 4 14 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 91 91 91 pu 2 2 2 4 2 4 2 4 2 4 2 4 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 2 2 2 2 2 2 2 D - D - D - D - D - D - - - - - - - D A A A A A A A A A A A A A A A A A A A A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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' 3 ot ac au au ag a c a a a u u au a aa a a a a a a a a a a a a a a a a a a c u a a ' c c u u g u c u a u a aa au a u c c c a ua u u u c u a c u c a 5 ga c ec u g c a c c u u g u c g c c u u g u c u u u a u a u a u a g u u g a u u c a ua a u u c u u ua a u u c u u u c a a u u u c u ca n u u c a u g c a c u u g g c c u u u c a u a g g au u u c c a ca g c c a c u a a a u a c u u u a c c a gg u u u c g u u u u c e a u c u a g c c u c u a c u u a u g c c c a u c c c c g g g u u u uq c f a f u f c f u f a f g f c f g c u g u c u g f g f g f u f u f eS U f C f A f U f C f U f u A f f G f u C f a f f u C G f f c A f f c C f u C f g f g f u U f f f f A A f U f U f U f G f G f G f U f d U f U f C f A f U f C f U f A f f C f f A U f f f C f f U A f f f f G f C f U f C f C f C f C f C f A f A f U f U f U f G f G f G f n U rt u U u U u C u Ac f U C U G C G A G U U C C A C A A C U U U G G a f f f f a f u f c f a f a a a f u c u g f a f a c f u f u f u f g f S Uu Uu Uu U U C u u u A c U f a U c U f g C g f g A f f u f f f f u c U c U c G Ug Uu U U U A f u g a u U U C U U u u U c u u es u n c u u u u u u c u a u c e u u c u u uu uu uu uu a c u u c u c au a c u c a u c g u u c ua gu a u g u c u u u c u u u u u u c S u u u u u c u u u c u u u u u u u u u u u c c a u u c u g c a a g a a c g a c c g a a a a c a c u g g u u a a g c c c a c u c u a u c u u u u u e 1 .2 1 .3 1 .4 1 .5 1 .6 1 .7 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . 1 . m 1 1 1 1 1 1 81 91 02 12 22 32 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 a 6 6 6 6 6 6 6 6 6 6 6 6 26 26 26 26 26 26 3 3 3 3 3 3 3 3 3 N 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 69 69 69 69 69 69 6 6 6 x e 1 l 4 14 14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 91 91 91 pu 2 2 2 4 2 4 2 4 2 4 2 4 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 - D - D - D - D - D - D - D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 2 2 2 2 2 2 2 2 2 2 2 D - D - D - D - D - D - - - - - - - D A A A A A A A A A A A A A A A A A A A A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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S n S N 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ' d u 3 ot a a a u u a c n o a C, ' 3 A A A A A A AC A A A ' uu u u ug r t d o A A A A A A U U G G U G U C G G U U A U 5 a ua uu u S na t' AA GA G G G U G U C G G G U U U C U A ec u c ne u c u c a g e u s a u c c u r t 5 A A A G G G U G U C a n S u g e s e U GC A C A G U U G c AC A A A G G G U G U A A A A G G G U G U U G U A A G U u e s n A A A G G G U G A C U G q a f a f c f a f i t n e U C A U U C A A A A G G G U U G U G U eS G f A f A f A f n A e uq C U U C A A A A G G G G U U U A G S A C U U C A A A A G G G C A A d A f G f A f G f e C A A G G A na U rt a A G G d f u f a f a f n e S C A a h t d GC C C U U A A G A C U U C A A A A A U U U G G C A C U U C A A A A G UA C U S U es a A U A e s n n c u a a o a G C G C A C U U C AC A G U U G A CA u n r t CC G C G C C AC CA UC U U AC UC U U G G n g a c u a e 6 S A CC G G U C G C G C A C U U A U G GA G e aa g a a cg aa S no e s A A C C G C G C A C U G C U C A S g a a u d ei i t n C fi i s e C A A C C G C GC C AC C G U G G U C CC S G C C A C A A C A C C G C G G C G A C A C U U G C G e . m 0 . 1 . 2 1 . d o 1 1 1 3 o P 8 9 0 1 2 3 4 5 6 7 8 8 9 0 4 5 a 1 1 1 1 m o n t 20 20 30 3 3 3 3 3 3 3 3 4 4 5 6 6 N 99 99 99 99 U d et 80 80 8 0 0 8 0 0 8 0 0 8 0 0 8 08 08 08 08 18 18 18 18 18 x 1 1 1 1 x 0 0 0 0 0 0 0 0 0 0 el 4 p 2 4 2 4 2 4 . 2 4 ag e l e 5 2 5 u - D - D - D - D e l u j pu ma - 2 5 D - 2 5 5 5 5 5 5 5 5 5 5 5 5 5 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - 2 D - D D A A A A ba no D N A A A A A A A A A A A A A A A A T C Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000236_0001
' A A 3 A A A A A A A A A A A A G C U U U U G U G U G A U U AC A A A A A A A A A G U G A U U UC U A A A C U A G A A A ot U U C U U U U G U G U G A U U C C A A A C U U A AC G ' A G U C U U U U G U U G U G U G A U U C A A A U U U C U 5 U U G U C U U U U U c G U G U G A UC U C AC A G G C A A e G A U G U C U U U U U U A G U C U U U U G U G U G A U UC U U U AC U A U G A AC n U U U G U G U G U C A U U C e G G U A U G U C U U U U C U U U U U G U G A C G C A C A u U U G U A U G U C U U U U U C U U U G G A U C A G C G A qe G G U G U A U G U U U G U A U C U U U G A U U G A U U U S A U G U G U U UA G U U G U G U C G U G A U G G G A C d A G U G U G U U U U U G G U A U G U C U G U G A A G A C G n G A G U G U U U U U C a U A A G U G U G U A U G U C U U U G U G U G A A U U G G U A U G U C U G U G U G A U G A rt C G A A G U U G U A U G U UC U U U G U G A G AC A C S A U G A A G U G U U G U A U G U UC U U U G U C A G C U es G C G A UC G A A GA U G G U G U A U G U U U U G A C G U G n A G A U G A A G U G U G U A U G UC U U A A G AC AC e S C C G G CA U G G AA G U U G U G U A U UC U U U A A U G A G G C A U C U G A A G A G U G U G U U A G U U C U U C C A C U G U A 66 76 86 96 07 17 27 37 47 57 67 77 87 97 08 18 28 38 48 58 68 7 3 4 1 4 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 81 12 1 3 3 3 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 28 28 2 2 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000237_0001
' 3 A A A A A A A A A A A A A A A A A A A A C A A A A A U A A ot A A C U G C U C A A G U U U C U G U G U A U A G U C C ' A G U A U G C U C A A G C U U C U C A 5 A A G A A U G C U C A A U C U U C U C U C G U A A G U A U C U A G A A C U e G A c G A A A A C A A U G C U C A G U C U U G A A U G C U C A G U C U U UC G U U G U AC U C G C G U AC UA U A n U A C UA U G A A U G C U AC A G U C UC U U UC C U U eu A G U G C U G A A U G C AC A G U U UC UC G U U G AC q A G C A C U G A A U G UC AC A G G U U C U UC G G A e C S C U G U A A C U G A A U U A A U G G A A C U G A A G C AC A A U UC U U G UC A C U U U C A GA U UC U C A G dn A A A G A G A A C U G A UA C U C A G U U UC U U C UC a U C C C A A A G C CC AC G A A CA U G U G A A G C U C AA G U U C A G A A C U A U G C U C A G A A U C UC G U rt G A U G U C A G A C G G AA UA U G C U C A G U U U U S U U C A U U C CC AC GA A A UC A A U G C U C A G C C e A C A G C U U C C G A A UC G G A A U G C U AC A G U U s C G A A CC CC U U U C AC G A A U G A A G C U A A C G n U U G AA C C U U C A G A CA UC G A U A U U G A G C C C A e S G A A C A U C U C C C C U C U U C C A G A A U C A U A U G U C U U A C 04 34 55 95 58 68 78 88 98 09 19 29 39 49 59 69 79 89 99 00 10 2 3 4 5 6 8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 03 03 0 0 0 0 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 38 38 3 3 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000238_0001
' 3 A ot C A C A A A A A A A A A A A A A A A A A A A A A A A A A A C A A A U G U U A C C A A A A C C G A A U G C U G C ' U C AC C A A U G U U A C C A A A A C C G A A U G C U G 5 U U C A CA A A U G U U A C C A A A A C C G A A U G C U ec G U U AC A C A G U C C A A A U G U U A C C A A A A C C G A A U G C A C A A U G U U A C C A A A A C C G A A U G ne U A G U C C A C u A U A U U C AC A A A U G U U A C C A A A A C C G A A U A C A A U G U U A C C A A A A C C G A A q C e A U U C A G U U C C A A G U U C A C A A U G U U A C C A A A A C C G A C A C A A U G U U A C C A A A A C C G S G U C A G U U C AC A C A A U G U U A CA C A A A A C C d G U U A U A U U C A A C A A U G U U C A CC AC A A A C n UC G C A G G U U CC AC A C A A U G U U A C AC A A A a U U A A U C U U C A A G C C A C A A U G U U A C AC A A rt U C U C U U U U A U C AC A C A A U G U U U A C AC A S UC U G G U AC A G U U U C AC A C A A U G U U U A C A e U s U C U UC U n G C G U C U A A U GA G U U C AC A A CA AC AA U G A U G U U A CC U C U U U C A G U U C G U U A e S A A G U U C G G U AC U C A G U C U U U C U G U A C U A A U G U U C A A C A A U G U U A G U U C C A C A A C A A C A A U A U G U 90 01 11 21 31 41 51 61 71 81 91 02 12 22 32 42 52 62 72 82 92 0 1 2 3 4 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 33 33 3 3 3 3 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 38 38 3 3 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000239_0001
' 3 A A A A A A A A A A A t G AC A A A A A A A A A A A A A A A o U U C A C A G A U U ' C U U C A C A G U U U G AC AA C A A C A U G U A A C A C U U C A C A A C A U A U A A C A 5 G C U U C A C A A U G A U G C A CA A A C C A A U A A C ec UC G C U U C A C G U U U U G C U U C A A U U U U U G AC A C U U G C A A A A A C A A C A C U A A C C C A A U A n G C U G C U U C C A U U U U G C A A A C AC AA CA A U e U G C U G C U U AC G A U U U U U G AC A A A A C A C A uq A U G C U G C U A G A U U U U e A A U G C U G C U C U U G C U C A A C AA CA S G A A U G C U G U A AC G A U U U U G A U C A A C A C C A AC G A U U U U U C A G C A A C d C G A A U G C U G U C A A G A U U U U U AA C U G C A A n C C G A A U G C ar A C C G A A U G U U U C C t A A C C G A A U C C U U A A C C G A U U U A A U C G U U G C A C G A U U A A U U U G C S A A A C C G A A G G C U U C A A G A U U A C U G C C A U U U U G U U C A C G A U C AA U U U U e A s C A A A C C G A U C n C A A A A C C G A U G C U U C A A G A G C U U U U U A G C U G C U U C C A G U C A A U U U e A C S U C A A A A C C A C A A A A C A A C A C A G C U G C G C U AC C A U U G A U A U G U C U G U C U U A C C A U U A C G A G U A U 63 73 83 93 04 14 24 34 54 74 84 94 05 15 25 35 45 55 65 75 85 9 0 1 2 3 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 53 63 6 6 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 38 38 3 3 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000240_0001
' 3 A ot U A ' C C A A A A A A A A A A A A A A A A A A A A A A A A A G A C C U U C C A A C A G C A U C A C C A A A G C C 5 A C C U A C C U U C C C G C A C C U U C AC A C A G C A U C A C C A A U G C C AC A C A G C A U C A C C A A U G ec A A U G C A C C U U C AC A C A G C A U C A C C A A U n A C CA U G C A C C U U U C A A C A G C A U C A C A A A e U A C C U G C AC C C U U C A A C A G C A U C AC C A A u A A A A C U G AC CA C U C CC A A C AC G C A U C C A q C U A C A C U G C C CC U U CC AC A A A G C A U A C C e A A A C A C U G AC A CC U U C AC A C A G C AC C A C S A C U A A A CA A C U G G C A C U U U C C A C A G U C A d C A C U A A CA AC C U G C C C U U C AC A C A G A U C n A A A A U AC C U G AC CA CC UC U C A A C AC C A U ar t AC C A A C A U A A C A C A U A A A A C C U A AC C C A UC G AC C U A CC UC U CC AC A G C A C A A A G C S G A A AC A C A U A A C A U G G C A C U U CC AC C A G es U C U G AC A A A C A C A U A A C C U G C A CC U U A U C A C A A AC AA C AC U A A AC C U G C A C U U C A C n U U G C A C A A AC U U A A AC C UC G C C C C U G C C U C A A e S U U U G A C A A A C A A A C A U A A A C A U A A C C U C U C C A C 56 66 76 86 96 07 17 27 37 47 57 67 77 87 97 08 18 28 38 48 58 6 7 8 9 0 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 83 83 8 8 9 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 38 38 3 3 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000241_0001
' 3 A A A A A A t U A A A A A A A A A A G A A A A A A A A A A A o A U G C ' U G U G U UC A U A A A C A C C A A A G A A G A C A C U G A A U U U U A U A UA A G U C U U G A U U A 5 U U C U U G C U A A A G G C U A AA A A U C U U G A U U e A C A G G U G A G A G U A U c U A U U U U A A A G U A A A U C U G C U G AC G A A G U A G U A A A G A A U C U G U n U U U CA C U U A A G A A C A U G G G A A G G A U C U A e AC U A G G CC G A A A A G C C G G U A A A U G A U C G uq A U U U U U U G A G A U G U A U U C G A A C A G A U U e UA U U U G C C C G G A A A U A C C U U G A U A A G A C S C U AC A C C G A G A A A G C U G CA C U G A A A A G U d C A U UC G G CA A G G A A G A U A UC C U G G A A A A n G C U U A U U UA CA G A U G U A A A A U C A U G A A G a G U AC A U C U G U C A A G U C A A A CA U A C U G A A rt U AC C U GC C UC C G G C G G A A A A C A U C U G A S C C G U UA UC G U C A G U A U U G A A AA G C U C U A e G G G AC U A G C U U A UC C G G G G A A A U A C U C G s A G U C U UC UC G C C C C A U U C C A G A A A A C U U n G C G U U C G U G UA C G G UC G G A U G U G G C A A A A A A A C C e S G U C G G A C A G A G U U C U C A A C U C U U C A A A A A U C 80 01 31 51 12 52 33 35 29 39 59 89 31 43 83 14 05 95 16 26 36 4 5 6 7 8 9 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 65 65 6 6 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 58 58 5 5 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000242_0001
' 3 A A A A A A A A A AC A A A A A A A A A A A A A A A A A A ot G A U G U G G A U A A A C U A A U U G U A U U U C U G G ' U G A U G U U G U A A G A A A G U G AA U A C A U U U G 5 A U U G A U G A U G A U G U A C A U AC G G AC C G U U A A U U G A C U U e G A A A c U U A U G A U G C A G A A A A A A G U C U A U A U U A A A U A G U G A A G A A A U C n U U U A U G G A G G U AC A G U C A U U G A A A A G A U e A U U U A UA U G G G G AC G A G U A G U G A G A A G A u G A U U U q A U A U G U A U A A U G GA U G U A U G A A G e U G A U U U U A A U U U A C A U GA C U G G A U A A A S C U G A U U U U A G U G U U G C G A A G C U U G A G A A d UA C UC G A UA U U C U G G U A A G G G C A A G U G A n U U UC G A U U C U U G U GA C A GA U G G G G A A U G a GA A A U U G G A U U C U U G A A G A G U G A A G G A U rt A G G A C UC G G CC U G U G A G G A U U G C A A G A S A AA A U UC U G A CC U G U C G A A G A G A C C G A G es G A A G A A UA U C U U A C U U U A U G G UA C U C A G A U G A A G A U U C U U C A U G U A G G A A C C U C A G n C G A AA GA G A G U C C U G C A A A G U C CC C CC A e S U U C U G A A A G U G U C A C C U C G U U U G A A G A C C U C U U C U C C 07 17 27 37 47 57 67 77 51 91 02 32 42 72 13 43 27 57 20 30 40 8 5 6 7 8 9 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 7 7 7 17 27 2 2 2 2 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 78 78 7 7 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000243_0001
' 3 A A A A A A A A ot U U U A U U G U U U A A A A A A A A A A A A A A A A A A U AC U G U U U U G A G A A A G G A C A G U A U ' G U C U U U A A G G U U C C U G U U A U A C C U C AC AA U G A G A A A G U AA U A 5 C G C U G G U C A A U U A U e C A A U G A C U C G c U G U A U A A UA CC U G G U U G U U G G A A G G n U U U A U U A A U A U G AC A U A A C G A e C U G A A A A C U C U U U U C C C A A U A U U A U U G u U C G G C C A A A C C U G A G G A G C G A A A A G A C U q A A A G A C U C C G C A e A U U C A AC U A A A U C U U U U U C U A G U A U G U U C C G A A A U A U G A U S G A A G UC G A A A A A C A U U U A G G UC A A G A A A G U A d A A G A G C G A A C A C U C U U G C A A G G A G U na A A U rt G A A A G G U U A U A A A C C G U C G U C A C A A U U UC G G C A G A A A U G U A G UC AC A A A C A G U G S U G G A U AC G G C U A A A A U U C U G A A C A U G G C U A U A G A U A U A G A A C U G U G CA A C A A G C es G A A C G C C GC CA U G A A C G U UA U A U C A U A U G G n A G G U C G G AC U G AA C U C U C G A A G A U U G e A U G U G GC A A U A U U G U AC G UC G CA G C U U U S G A G U A U U U U A U C G G C A A C G A C U C G U U U G U G U A A C A U U 13 23 53 94 96 07 18 48 58 78 88 98 29 79 10 40 70 80 41 51 71 8 2 3 8 0 1 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 18 28 2 4 5 5 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 88 88 8 8 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000244_0001
' 3 A A t A A A A A A A A A A A A A G A A A A A A A A A A A A A o C A A U A U A U U U G AA U A U C G U G U A G A U G U U ' U C U A G C U A U U U G A U A G U U U C G A G A U G U 5 A AA A U U A C U A U U A G U A A C A G U G G A G U U G ec U U A U G A C A G U U U U G A UC A U G A U A G A C A G A G U G G A A U U A U A U U G A U A A U G A U G G G A U n A UA C U A A U G C G U U U U G A C G C U U A U G A G A e G C C C U U A U A C A A U U U G G U G G G A U A U G A G u U U U C CA A U G A A UC A U U U A U U A C A U A G G A qe C A C UC U A U UA G A U A C A G A U U U C A U U G G S U U G G G U U A UA U G CA U G A U U A C U U C A A U G d A A G U C U U U A U A U G C G G A A AC G C U U C U A U n U A G G A C CC UC U U U A A A U G A C U C C U U A U A a G G A G U UC C C U UA G G U G U UC U AC C C U C A U rt G U C AC A UC U C UC U A U C C G U A AC C C U C A S U C AA A U G C U C U U A C G U C UC A AC A A C U U C e C U A U G G G C U CC U A U A G C C G C G C CA A C U U s G A G G CC G G G G C U UC U A G U G A C U G C C C C U n G U U U AC G G G G C C U U U G C G C G G G AC A C G C C U G G C C C e S U G A C A U C A A C A G G G U C C U G G C U A U U A A C C A 25 75 96 07 37 57 67 77 87 97 08 18 28 48 11 61 81 52 93 24 06 1 2 3 4 5 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 69 69 6 6 6 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 98 98 9 9 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 8 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000245_0001
' 3 A A A A ot G G U A A A A A A A A A A A A A A A A A A A A A A A A G U CA AC CA A A A G G A U U A U G C U C A U C G A ' U G 5 U U G G U A C CA AC A A G A C C U UA U U G C U C A U C G U U A C A A A A U C G C U A U U G C U C A U C e G U G G C c U G U U G G U A C U A A G A U G C U A U U G C U C A U G G U A A C CA G A C U G C U A U U G C U C A n U U G U U U U G G U C A C A A C C U G C U A U U G C U C e A U G U U U U G G A U A AC U A C C U G C U A U U G C U u G A U G A U G U U G G G U A G A A C C U G C U A U U G C qe A G G A U U G U U A G G C G A A A C C U G C U A U U G S G A A G UA U U G U G U G A A U A A A C C U G C U A U U d G G G A C A G A U U G U U U C G U A A A C C U G C U A U n U G G G A G A U U U G U G A G G U A A A CA C U G C U A a A U A U A G A U C U G G C A G G U A A C A A CC U G C U rt U A A G G G A G A U U U U A A A G G U A A A A C U G C S A U U U G G G A G C A U U U C A A G G U C U A A A C U G e C A s U C A A U C U A U G G A G G A G G AC C A A G G U A A C A U G G U A A C U G U G A AC C A A G G G U A A C C n U U U AC U U A U G U G A U U U A AC C A A G G U A A C e S C C U C U U A C A U A U A G G A U G U A A C C A A C A A G A G G U A A A A 76 86 96 07 17 27 37 47 57 77 87 97 28 58 88 98 09 19 29 39 49 5 6 7 8 9 0 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 99 9 9 9 0 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 98 98 9 0 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 8 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000246_0001
' 3 A A A A A A A A A A A A A A A A A A A A A A t G AA A A A A o G A C A U A A A U C A A A G A A U A A G U A U A A CA U ' A G A C A U A A A U C A A A G A A U U U G G 5 G A G A C A U A A A U C A A A G A A A G AA A CA C G U G U U A A e C G A G A C A A A c U C G A G A C U A A A A U C A A A A U C A A G A A A G C A n A U C G G A G U G G G U G A A AA GA U U G e C A U C A G G A A G AC UA A A A u U A A A U C A A A A U C A AA U A G A AA A G U G G U A AA GA U U G q U C A e C U C UA C G A A C A U A A A A U C A S G C U C U C G U G A A A A G C G A C A U A A A A U C G C U AC U A G A C A U A A A A U UC U G A A A A U U G A A A A A A G d U G n U U G C U AC UA GC A G A C A U A A A A A G U G A A A AA a A U U G C C G A G A C A U A A A A A GA G rt U A U U G UC U U U G A A A A C G A G A C A U A A A U U A G U G A A S C U A UA U G C C UA C G A G A CA A U A G UC U A G G A U U G U C U C GC A GA C AC U U UC A U U G e G C U s U G U C A U G G A A U U U G C U A U n C U G U C A U C G A G A C G A U C U U A G U G C U A U C C G A U A U C A G e G C U C A U G A U U U S C A U G C C A C C U C U G U C U U U G U C U A C U C A A U G C G G G A U A U C U U A A A A A U A U C U U 10 20 30 40 50 60 70 80 90 01 11 21 31 41 51 61 71 81 82 53 63 7 8 9 0 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 30 3 4 4 4 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 09 09 0 0 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 9 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000247_0001
' 3 A A A A t G U A A A A A A A A A A A A A A A A A A A A A A A o U G C ' U U G C U G C UA U U A C A G U U AC U A C U UA CA U U U U G A U A C A G A C A 5 C A C A U G A U A A AC A U U G U G CC A AC A C A A C C U A G G A U G A U A ec A C n A A U U G U G U A A C C A A C A A A A G U U G A U G A UA U A U U G U A U A U G A C G C A U A U U G A U G A e G A A C U U G C U U A U A A C A U AA A C U U G A U G G u U G A AA C U A U U AC C G A U G C C A G C U U G A U U qe G U G A A S U G U G A C U G A A U A U G A C C A G U G C U U G A A A A UC UA U G A U C U U U U U U A U G C U U G G U A A G C U UA A A C C A C A A A C A A U G C U U U d G G U G n A G A G AC A U G C A U A U A G U A A A U G C UC a A GA U G U U A U A G U A A C C G U C G A A A U G UC rt AA A G U G G AC A A U C A A A A G U G U G G A AC U A A A A G UC G A A A U G G A U C C C G G C U G A A A U S A A AA A G U G A U G G U U G A U U U U CC U G A A A U es G A A A A G A G n G A A A U G U A U A U U A C G CC U A C U CC U G A A A C U G A A U A A G U A G A A A C U G C A e U G A A G G A U U C C U G S G A A G U G A U C G C G G A G G C C G A A A A A G G U C U G U A U U A C A U A U U U G G U C U C C U C U 34 44 54 64 74 84 05 97 78 88 09 49 69 79 40 60 12 23 55 95 06 1 2 3 4 5 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 61 61 6 6 6 6 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 19 19 1 1 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 9 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000248_0001
' 3 A A A A A A A A A A A A A A A A A A A ot U G A A G U G A A U A C U A U G A G U AC A A A C A A A A G A C UC U C A A C ' A U G A U G U G A A U U C C C U G GA U U C U U A A A 5 AC A U G A U G U G A A A U U U U U G C U G U C U C A A A A A U A A U G U G A U A U G U U A G C U A G U C U C A ec C A A G A A U G U G A U CC G C U U G C G A G U U U C n UA A C A AC U G A A U G U A A U U UC U U G A CC U U e U A A U G A A U G G A U U A UA U UC U U U G GA CC U u G A U q U G A U AC A U G A A U U G GA G U A U U U U G U C U C eS A A A A A U G A A G U A U U C G U A A U A UC U G U G U G A U G U CA AC AA U G A d A G U G U C U G A A U U U A U C U A G U U G A G U A A C U A U G A A A U C G A n U G A a U U G A G UA U A AC A U A A U U U U G A UA A U U G rt C U U G U G A U A A A G A U A G U G A UA U UA C U U U A G A C U A AC U G C U U G G U U G A A U A U U S G C U U U e U G C U G A U G A U A A U UA C AA U G A A C U U C s A U A A A U A U G U U U U G C U G n G U G A U C A U A G A A U G U G A A A U C A G A G A A C G A G U G A A U e A A S G A U U A A C U U G UA G A AA CA U G AA U G U G U A A U A G U C U U G U A U U A U G G A A U A U G A A U A U G U A A U A U 76 86 96 07 17 27 37 47 57 67 77 97 08 38 48 58 68 78 88 98 09 1 2 3 4 5 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 91 91 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 19 19 1 1 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 9 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000249_0001
' 3 A A A A A A A A A A A AA A A A A A A A A A A A A A A A A ot G A C G A U G U G G U G U U U A A U U U U G A A U G CA A U G G U G C A U G G U G ' G A U C U G U A U U A U C 5 A C G C U C U U U U A C G C U U U A U A U G A C AC U G U e A A C C G U C A G A A U G G c AC A A UA U U A U U U G G A A U U U U U G G G U C C U A U U A U G A C A G A C U G n A U C A C U A U C U U G U U G C C U A U U A U G A AC U e U AC A C U A U C C G U C U G C U U A U U A UA U G A A u U q G C U U A C U U G C U C C eS C UC U U U U A U C A U U G U U U A U C A A U G A U C U C G A U U C C U U A U U U A U G C C C G A AC U U A A C U G A C U A U C A U G C A A G CC U U A U U A U U A U d G U C U U A C U C U n A U U A a A G U A G A C U C U U U A A C rt G A C A G G U G A A C U U G C C U U U A U U G A C C U G C U G U G A U A C A U U G C C U U C U A A AC U G A CC G U A A A U U G C C U U S U G A G C U A C G G A A G U CC A A U U G C G C UC U A e U s U U U AA A G C G C C C G A G U G U U G U G A G A C A A U U U G C U U C U C A G G G A U A CC A A U U G CC U n U e C U U C G A U C A A U C S U A U U C U A A C C A U G A A G U A U G C A G C G A G U G U G G G A G G A G U C A C C A C A A U A U U G 79 89 99 90 31 41 51 71 81 62 73 58 68 88 98 09 19 29 39 49 59 6 7 8 9 0 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 92 92 9 9 0 0 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 29 29 3 3 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 9 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000250_0001
' 3 A A A A A A A A A A A A A AA AC A A A A A A A A A A A A ot C U U C A U U A U U G U G G A CC A G A G U U U U A U U A ' C C U U C 5 G C C U U G G A U U U G U U G C C U G U G G U U U G U G A CC AC G A G U U A C C U A U A G U G A C A A A G U U G A C U G U U U G U G A CC G A A G G A C G ec G U G C C A G C U U G U U U U G G A AC G A A A A G G n G G U G C A G A G U U G U U U A U G U G C C G A A G A U e U G G U G G A G G G U U G U U A U U G U A C C G C U A U u A U G G U A A U A G G U U G U A G U U G G A C C C G G G q C AC UA G G G A G A A G G U U U G U U U G A C A U U U eS A G A C U G U G U A A A G G U U U U U G U G A G U G GA d G A AC U A A n U A U G A A A G G U U G G U U G U G C G U G U G A AC G A G A A U A A A G G U U G U U G U U A U a A A U G A G U G G A G G A A A G G U U U U U G U A U A G rt U S U U A U UA G A U U A G A AA AA G G U U G U U U U C A GC e A U U GA A A U G A G U A A U G A G A AA G G U A G G U G U U G G A G s U U A A A U n U U A U A G A A A G A U G U G C U U GA G G G A AA U G G A G A A G U U G A G UA e U A G A U A A A G G U S C C U C U U U A U A A G A A G G A G A A G U U G A A G G A A A A U A U G G A G A A G A G G U A C G A U U 20 30 40 50 60 43 73 83 04 14 24 34 44 54 64 74 84 94 05 15 25 3 4 5 4 6 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 53 53 6 9 9 1 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 39 39 3 4 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 9 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000251_0001
' 3 A A A A A A A A A A A A A A A A A A A A A A U A A A A A U ot U A A A C U AC A A A A U A U A A U U G U U G C U U U U ' U U A A A C U A A A U U G A U A A U U G G A G U U U U 5 A U U G G A C AC G U C U G A U A A A U U U A U AC U U ec U A U A A G U A A A G G U U A A U A U G A U A U A A A U U C A A n C G A U G A A A U G A U G A U U U U C A C U CC C eu U G U U C G U A G U G A A G C U U U G A U G A AA U U U C U U U C A U G U U A C A U G A U G A U A C U U U U G q U U C A U A U U G G G G A A U G A U A A A U C U U U U e C U U U U U U U U U U A U A A U G A G UA U C U C C U U S U C U GC A U CA U U A U C U A A U G U G A U C G U C U d C U C U U A A C A U G G A C C U A A U A G AA U A U U U n G C U a U G C U G U C A CA U U C G C CC U A A G UA U A A U U rt C C C G U C A U A UC A G G CC U A A A AC UC U G S A U G U U C C U C U A A A A C U U G G G A A G UC U A C U C U U C U C U UC AC A A G C CC A U A G UC A A es U A C A A G UC U A U C A C A C U G AC A A G A A U n C A A G U A A A A A U U GA A C A U C U A U C G A C A A C G e C U U S G C C U C C C A A G U C U U A G A U U C A A A A U A C G C A A A U C U G A C A C UC U A A A A U C UA U A G C C U A A U C G 94 05 15 35 65 75 67 77 87 08 49 59 30 40 50 60 70 80 01 11 21 1 2 9 4 5 6 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 35 35 3 4 4 4 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 59 59 5 5 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 9 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000252_0001
' 3 AC A A ot G U U A A A A A U U U A U U A A A A A A A A A A A A A A A A A A A U A ' U A U U A G C U C A U U C A C A C A U G A U G A A C U U U U U U A G C A C A U G A U G A A 5 U C U A U A U U U A U A U GA G A G C A C A U A A U G A ec U C n U G U U U A U G U U C G A U C C U U A U U U U U U U G A A A A G C A C A G A A U G A GA A G C A C U G A A U e U U A A A U u G U C C A A A C U U U U C C A G A G C A A U G A A A U U U U C A C U U U A C C AA G A G C C A U G A q U U C U G A A C U A U U U G C A C C A G A G A C A U G e A U G AC U G U C C A C U G A U A A C C A G A C A C A U S A U U G A G A C A U C U C A A G A A C AC A G G C A C A d G G U AC U A A U A C U A U A G U G A A C A A A G C A C n U U U A CA A A C C C C A A A U U G A A CC A G A G C A a G A U G G A A U A A U C A AC C U U G A A CC A G A G C rt U A U A U G CC C C A C U A C A C U U G A A A A G A G S A G G AA CA G U U C A C A C U A U A C U A CC A A G A es U U U U G AC A A A AC U A A A A U U A C U G G A C A A G U G A A A A C C C C C A U U C C G U U U A C A A C A A n U A G A G G U A GA C U UC U U U U U A U U U U U C G A C C A e S U C A G A U A A C U A C A A C C C U C U U A U A U C U G A A C A C C 15 55 85 39 69 99 92 47 00 31 81 02 12 97 10 21 31 41 51 61 71 8 9 0 1 2 3 5 5 5 5 5 5 6 6 7 7 7 7 7 7 8 8 8 8 8 8 8 18 18 2 2 2 2 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 89 89 8 8 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 9 5 0 9 5 05 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000253_0001
' 3 A A A AC A A A A A A t U A A A A A A A A A A A A A A A A A o A G U A A U U U A A AA A A U A G G G A G A G U G G U U ' C A U A U A U G U 5 A C A A C U C G G C A AA A A G C C U G A G A G U G C U U U A A A C U A G U G A G A G U A U ec A A C G A A A G C U A U A G U G A U G AA U U A A U U A U A A C G U G A G A G U C A A U G A C GC U G A G A C C n U G A U A U U U G U A C U U U U A C A A A G U G A G A U eu A U AA A C A C U G U U U A U U G A C A C G U G A CC U q A A A G G C G C U U U U e G A C U A A G U U U A A UC U U UA A A G A U U C G AA C G U G U G A U C A C G U A U S U G G G G A A A U G G A UC U A U A A U GC A A C G A U d A U U A G C A U C U G A U U U U U A U A U GC A A C U C n C A U C U G C U G GA G G C U U A A U A U GC A A A A A a A C A rt C A G U U U A G A U A U G G C A A G U C A UA U A U A GC G C A A U UC A G C A G C A U A A U A U C U A S G C A A U A A AC U A G C G A A U A A U UA A UA U U U e A G s G A A U AC A A U G A U G C U G U U A G AC AA A U U U A A U U A C C A UA U U A G U C U U A A A UA U U A n A GA G U U G A U U C A C C U U A C A U U U U A U A U C e S A C A G U A U A G A G U G A G U U U U C A A C A U C U U U A A U A U G C A 42 52 34 26 59 69 99 30 80 61 92 03 13 23 65 26 56 76 86 96 07 1 2 3 4 8 9 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 79 79 7 7 0 5 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 99 99 0 0 x el e 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 05 0 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000254_0001
' 3 A A A A A A A A A t U A A A A A A A A A A A A A A A A A A A o C U U C A U U C ' C C U U C A U U U A AC A A A AC A A G A G A U A C C U G A C A A A A G A G A U A C C U G A C 5 U C C U U C A U U U U A C AA A A G A G A U A C C U G A ec U U C G U U U U U C A C U U U C U U U U A C AA A A G A G A U A C C U G U AC U U U A C A A A G A G A U A C C U n U G U C C U U AC U A A U U U A C A A A G A G A U A C C e U U G U C C U U U U C A U U U A AC A A A G A G A U A C uq C U U U U C C e A C U G U U C UC U C U U U C A U U U A AC A A A A A G A G A U A U U C A U A C A A A G A G A U S A A C U G U U U CC U U U A C U U C A A C A A A G A G A d A A A U U G U U U C UC U U C UA U U U A C A A A G A G n U A A C U U G U G U C C U U U C U U U U A AC A A A G A a C U A A C U U U U U C C U U U AC U U U A AC AA A A G rt A C U A A C U U G U U C C U U AC U U U U A C AA A A S CC A C A A A C U U G U A A G U U C C C A U U U A C A A e A CC A U A AA A CA U U G U U C UC U U U C A U A C A s n A A C C U A A A C U U U G U U C UC U U C U U U U A AC e G A C S U G A A C U A C C A C C A A A U C U A C U U G U U C U U U U AC U U U A A A A C A U C U U G U U C C U C U U U A C U A U U U 67 77 87 97 08 18 28 38 48 58 68 78 88 98 09 19 29 39 49 59 69 7 8 9 0 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 90 90 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 10 1 1 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000255_0001
' 3 A A o A A A A A A A A A A A A A A A A A A A A A A A A A t U C C C U G U A A A A G A A U U A U G C U C CC U A U U U U ' A U C U A U C C A U A U A C U U G G U U C U 5 C A U G A A C A U A U A G A C U CC U A A C U U A G U U C C A A U ec A C A U G A C A U A U C G G A A U C A G U C G A C U U A U U U U U U A U U A C U G U A U UC A C A U U U U C U C ne U G A A u C U G A G G U G A A A A A G U A A C U G G U A C U C A A C U U U U G G A U CC C G A G C U A G C U U U G q C C U G C G A A A U U U A U U U U U G U G e A C C A A U A G A C U G G A C G C U A G U U U U G U U A U G U UC A S U A C G C C G UC G U C U U G G C U C U A G U C C UA G d A U A U AC AC A C U A U U G U A G C C C U A G G G C A n G A U C A G U A A A A C U G U A A C CC A U A A U U U U a A G A A rt G A G C U G U G A C A U U C U U U A U A U U G C AC U S A G A A G A C U C AC A C U G C CC U G A A A A U G U G A A U G C A U C A U UC A G U U U U G A A U U G es AA A G A A A A A AA G G U AC A U A C UC U U G G A U GA G n A A A G G U U C G A U G G C A U U U UC A A CC U U A A U e C A A A U C U U U S C A A C A A U C C C U G G G A A U U G U A G C C A G U A G G G C G C G A C C U C U C C C C U C U G G A U 30 40 50 11 21 32 62 53 46 77 87 08 28 48 90 01 91 32 42 03 13 2 3 5 3 7 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 32 32 3 4 6 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 20 2 2 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000256_0001
' 3 A o U A A t U G A A A U A A G G A AC AC AC A G A A C A AC AA AA AA A A A A A A A A A A U U AA C U C A G AA A '5 GA U C U G G A C C C G C A C A A A A U U C U C A G AA U G A A U G G A C C C G C A C A A A G GA A U C U C A ec G n U G U A U U A A A UA G G A C C C G C A C A A U G U A A U C U C A A U G G A C C C G C A C A C A A A A U C U C e U u A G U C UA U AA U G G A C C C G C A C U C A A A A U C U U U U C A A U G G A CA C C G C A A U C A A A A U C q U A A U C U A e A U U A A U A A U G G C U A A U G G CA CC C G C U C A C A A A A U A CC C G A U C A C A A A A S C A U G A A U AC U A A U G G G A C C C U G C A C A A A d G U G A G A A U AC U A A U G G C CC C U C G C A C A A n G AC A C A G A A A U A A U G A G A A U CC C G C A C A ar t AA G G G C A G A UA C A U A A U G G A U A CC C G C A C G U G C A G A U C A U A A U G G G G A CC CC G C A S G A U U U G C A G A U C AC U A A A U A U G G A C C G C es A U A U U U U G C G A U U U U G A A A U A C G A A U C UA A U C A C U A A C A UC A UA G G A CC C G G G A C C n U A U A A U U U U G C G A A G A U AC U G A U G G CA C e S U U A C U A U U U U G A C A G A A U A U A C U A U A U U A A U A U G G C A 58 78 39 30 40 50 60 70 80 90 01 11 21 31 41 51 61 71 81 91 02 1 2 3 4 5 6 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 23 23 2 2 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 30 3 3 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000257_0001
' 3 A A A A A A A A A A A A A A ot A A A A A A A A A A A A A U C U C A A U A U C U G U G A C A U U U U U U C U C U U GA ' C C 5 A C AC A A U C U G U G A C A U U U U U U U C U AC A A A C C UA U A U C U U U G A C A U U U U U U C U C UA ec C G U U G A C A U U U U G A A C C A U A U U U G U U G A C A U U U U U U C U C n A G A A C C A U A U U U U C U e C A G A A C C A U A C U G U U G A C A U U u U C A G A A C C A U U C U G U U G A C AC U U U U U U C A U U U U U U q C U C A G A A C C A A U C U G U U G A e U C U C A G A A C C U A U C U G U U G A C A U U U U U S A U C U C A G A A U G A C A U U U U A C A U A U C U G U U U G A C A U U U d A A U C U C A G A n A A A U C U C A A CC A U A U C U G G U U G A C A U U ar t AC A A A U C U C GA A A A A U C U C G A C A U A U C U G U U G A C A U A A CA CC A U A U C UC G U U G A C A S A C A A A A U C U C G A A C A U A U UC U G U U G A C U C U A A C CC A U A U U C U G U U G A es C A C A G C A n C G C C A A A C C G G A A A A A A A A CC A U A A U C U G U U G A UA U AC A A A C A U A U C U G U U e C C A S C C C C G C G A C C A A A C A A A U A A C C G A U U C U A C G A C A G A C A C AC UA U A U C U G U A C C A U A U C U G 72 82 92 03 13 23 33 43 53 63 73 83 93 04 14 24 34 44 54 64 74 8 9 0 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 43 43 5 5 5 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 30 3 3 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000258_0001
' 3 A A A A A A A A o G A AA A A A A A A A A A A A A A A A A A t A G U U A A G U C A U G A C A C U U U A A C A A A C A U AA ' A G U U A U U G G U U C A A U U U G AA CA AC CA UC U U A U C A A A C U G G U U A A U 5 A U A C A C A U U A U C A A C U U U UC G A A G A C G A U A A U C A A A U ec A n C A U G A U U U U U A C A A A G A U A A U C A A UC U C A U C U U G A A G U U G U C G A A A G U U A A U C A e C U C A A U G C U U U U U U C U G A A A A U U A A U C U uq U A U C G G A G A U U A U U C U C A A G A U U A A U G U eS U U A U U A U A U U G U U A A A A U U U C C C A A G A U U A A A A A G U UA G U U U U U U C C A A G A U U A A d U A A U A U C U UA A U G A G U U U U C A A A G A U U A na U A A A rt U G A A U C A C U A U U A U U A U U U C U U C A A A G A U A A U A G G U A A G U AC C U C C A A A G A G S A A G C U A G G C U U A A U U A U G C U G C U U A U G A A C U C C A A A G U U AA A U A C AC U U C C A A A C es A A n G C U A AC G U G AC U A A G A A A C U U C CC AC A U U A U U C A G A U A A G AC U U U A A A C U U C A G e U C C U A S U U U C U A C U A U A G A A G U U U G G C U U UC G G C A C U U U CC U A G A C U A U U U C U A A C A A C C A U C U U U 45 46 56 66 96 07 17 37 87 08 18 28 38 48 58 68 59 69 79 89 99 0 1 2 3 4 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 04 04 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 40 40 4 4 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000259_0001
' 3 A A AA A A A A A A A A A A A A A A A A A A A A A A A A A ot C U A ' U U U A A C A U A C A C G A A A A U U A A U U A A U U GA U U A C A U A C A A G A A A U U G A U U A A A 5 G UC A A G U A C A U A CA C A G A A U U A C C G C A A e U U c A C U G U G U A C A U A U A C A G A U G A A U U C C A A U U AA U U G U A C AC A G A C A G U A U C G A C C n A G G U U U U G U A A C A G A C A AA U U U A A AC A G eu A U UC U C U U U G U U A A A G A C A A U U A A A AC A q G A UA U U C C U U U G G U A A A G A G AA G A G A A C e U AA A U U C C U U U G A A A A G G G UA U A U U A A S C A A G C U U C C U U U U G A A A A A A G G GA A U U A d U G A U U U U U C C U U U A G A A A A A UA A A A G U A n G G AA G A U U U C C UC A A G G AA G A A A U C U G G a U rt U C U A U U A U U U C C UC A AA A A G G A G U U AC S U U C A A C U e A G A C U A U U U C C U C G A C U A U U U A G A A G A A A A A G G U U U G U A G A A A C AA GA A s C G G A U U U UC A U C CC U A U U U U A GA G A G A A AC U A G G U C CC U A U U U U A G G G A GA C G G U n U e U G C U A C U U U A U G S U A A C U A G U C G U C A A G G U C C C C U C U A C C U A A A U C U U G G G A G U A C G A A A A U A G C 92 23 33 53 73 56 66 76 86 96 07 17 27 19 29 39 49 59 91 02 42 6 4 6 4 5 7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 25 55 6 7 7 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 50 5 5 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000260_0001
' 3 A A A A A A A A A A A A A A A A A A A A A C A A A G A A A ot A U U U C U U A C A A U A U U C U AC U U A A U G C C U ' C A A U U C C U C C G A U U U G U U 5 A G U A U C U C A U U G A U U U CC U AC U UC U U G C C C A A U U G C C e A c A A A U A U C U U C A U G G U U U G A U U C C C G C A U A G U G UC U C U A A A U G C U C U U U G A G U A A U n G e A A A A G A A U C U U U C A U A U U A G U U U C A U G C U C U A U U A UA A C U C A U G U U C C U A A U AA U u C q A A U A A A U C G C U G A A e U C A A U G U A U G U U G C U A G A U U A A A S A G A A A A A U U A U G C U G U U U G C U U A A A U A A A U G A U A G A U U U C G A U U A d U A C A A U G A A n G C G C A A A G U G A U C AC G U G U U G A A A U A U U a G A r A G C A U A A A G A t C C A A UC U A A U U U G U A C A U A G A UC AC U G U U AC G G A C A S A AA C A G A C A A U G A A G G G G A A G U A A U G A C A AC C A A A U GA A A U UC U G U A U C G A G G A e G G A s A n C AC A AA A G A A U G C G A A A A C AC A A A AC G A UA G AC A U G A A A G G G A U G A A G C U G G e A S U U G A A C G A C A G A G A A C G A G U A A A A C U G U C G A A A G U UC AC G G A U G A A A G U U A U U A U C A G A A A U A U 00 90 11 21 31 51 71 81 02 75 95 06 16 56 66 86 17 37 57 67 87 6 9 4 8 9 0 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 07 07 1 2 2 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 70 70 7 7 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000261_0001
' 3 A A A A A A A A A A A A A A A A t G A A A A A A A A A A A A o U A C A A A U C U U U A G UA U U A G U U C C C U A G A ' U C 5 C A U C A A A U C U U A U U C A A A U U U G A U U A U U C C C A U C U U A G G GA A U U A C A U U C U U A e C c C U C U U C A A A A U A C G G A A U C U G U A A U C C U U n G U CC C U U C A A A A A A C G G U AA U A C A U C C U e U U C C U U C A U A A A A C A A UA A UC C C U U C C u A A G C C C U U C A U U A A A GC G U C C UC C A U U C qe U G U G C C C U U U A G U A A A A U G C S A A A U G C CC C U C U G G U A A G U U U U A U C A U U C C A C C C A U A G U A U G C d C A U A U G C CC U C A U U G G U A C U G G U A A A A G A A U C C A A G CA A U C AA CA U C C n A A A A U A U G C A A U U U G G A A A A A U C A C U C a U A A A A U A U G A A UC U U U G U G U C U U U A A C U rt U A U A A A U A U C A A C C A U G A U U U U U C A A C S AC U U U A A AA U A U C U A C U U G A U U U U C AA A es A G A n G G C U U A A A A U U U U U A UC U U U A A U U U A A U U U C A U A AA C U U U A U U U UC U U U U U AC e G U G CA A U U A CC C U C U U U A U G GA U U UC U U S G U C A U C U U U U U U G G A C A U U G C A U U U U C G G U G U C U U U U 13 33 43 53 63 73 83 93 04 74 84 26 36 46 56 66 86 17 87 58 59 5 6 7 8 9 0 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 18 18 1 1 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 80 8 8 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000262_0001
' 3 A A A A A A A A A A A A A A A A AA A A A A AC A A A A A ot A C A U U C U U C U A C U G A A U G A U U CC A G A C ' A A C A U U C U U C U A C U G A A A G A A UA U C A G A 5 G A A C A U U C U U C U A C U CC G G A G G C G A A A G ec A U G A A C A U U C U U C U A C A G A A C A U U C U U C U A U U U G A U G UA CC A A A G C U G A UA U U CC A ne U u C U A G A A C A U U C U U C U U U A G A A C A U U C U U C A A C U G G G A A U CC U G A UC A C A A G UA G A q C C U U A G A A C A e U C C U U A G A A C U U C U UA A U A G U G A G U G A S U U C C U U A G A A A A C U U C UC U U C U A A U U C U G A A U d A U U C C U U A G U GA A UC U CA UC G G A n C A U U C C U U A G A C A A U U G U U U A UC G A G UA a C C A U U C C A G A C AC A U U C U UC U A UC G A C U U U A G A A C UC C U U C U C U A U G rt U C C A U U U CC U U A A A A A G AC U U U C U C G A S C U C C A U CC UC U U G G A U A A U UC U U C A UC G e AA C U C CC A U U C C A A G UC A CA AC C U U U A U s A C U C A U U n C U U U U C A C C U C U A U C G A A U C C e C A A C U C A U U U U U A S U A C A A A A C A U C U C C A C U U C U A A A U U C C U C U U C U U G A A G A C U U C U C A U U U U U C 12 22 32 42 52 62 72 82 92 03 13 23 33 43 53 63 73 83 93 04 34 4 5 6 7 8 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 48 48 4 4 4 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 80 8 8 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000263_0001
' 3 A A A A A A A A A A A A A A G C A A A A A A A A A A A A A ot C A A U U G G U A C U U A U G A A U G A A U G U G C A U ' C C A U U U G G U A C UC U AA G U U U A U G A G C AC 5 A C C C U U U G G U A A UC UA AA G G G C A U U A G G A C U C U U U G G U U U A C U U A A A U A CA A U U A G ec A G A U U C U U U G G A A U G U A U A A A U C G U U A n A A G G U U C U U U G U U G G U C A U G A G U A U G U U e CC A A U G U U C U U U G G G UC U A U A GA U A U G U u C A G U G U U C U U U U G U G U U U A U U GA CA A U G q UA C CC A G U G U U C U U U U U C GA U A A CA AC U eS G UA U A A G U G U U C U U U d A U G U U C U U C U G C A AC G U G UA U G U A AC n U A A A G C U A C U G A U G U A a A G G A A A A G U G U U U C U A U U U G G A GA G U rt G U C A A A A G U G U A U U G U C U A A G A U UA U A G S A A UA C C A A A A G U G U G G A C U U AA A G A U A G GA G A C C A A A A G U G G U G U A U U A A A G G A U es U n C G A G A CA CC A A A A G U U U U G U C G UA U A U U G A G A G C G A C A A A AA G U U G G G U U A U G U G e A U S U C A A A U C C C A C A A G A C A G C A A A C C A C A A A A G A A C U G U A A U U C U C U U A AA GA G U U U A C U U A A A G 05 15 25 26 36 46 56 66 76 86 96 07 17 27 87 97 18 28 78 88 19 2 3 6 7 8 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 98 98 9 9 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 80 80 8 8 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000264_0001
' 3 A A A A A A A G A A G A A A A A A A A A A A A A A A A A A ot A U A A G U A AA C U A U U G A UA A A U C U C U G A U ' U A U A U G G A UA G C U A U U G 5 AC U A U A U A U A U G C U A U U G UA A U C G C A A U G U U U G A C U U G ec G A A C U A A A A U U A A G G A A A A U G C U A U U G A U A U C A C U U U A AA U G C U A U U G C A A G U A C U n G C U A A A U U A G U A A U G C U A U U G A A U C U A C e U G C U A U G A G U AA A U G C UC A U U G A A G C U A u U A GA AC U U U A A G AA AA U G U A A A G A U G C U q G U U eS U U G A U A C U G G A A A A U G C U A A A A A U G C A G U A d C U G U G A A U G A GA U A U G C U A A A A A G A A U G n A A U A U G G U G A G U A AA U G G U A A A A A U C U G A A G A G A U G A A G A A U A G U A G A A A a U A AC U U U G A G A U A G U G A A U A A A G A A G A A rt G U A AC G U U G A G A G A GA G U A A C A U A A G A S A G U A U G AA U A A G G U G A A A G U AA C G A A A G es U A A n A G U C U U U G A A A G U U G A A U G A A U A AA AA U A G A AC A U U G A G A G G A G A UA A CA G U A e G A A G A G U A C U A G U S U G U A U A U U G A A U G A A A U A U G U U A U A U U U G A A G A G G A G U G A A U A A U C A A C G A G 00 10 20 30 50 60 61 52 72 82 92 03 13 23 33 43 53 63 73 04 08 1 3 4 5 6 7 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 89 89 8 8 8 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 90 90 9 9 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 0 5 1 0 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000265_0001
' 3 A A A A A A A t G A A A A A A A A A A A A A A A A A A A A A o U U C C G G AA CA C C U G C U A U A A C A C A G C U U ' U U A U U G G G A C C C A U C U A U A G C A C A G C U 5 A U U A G U A U G G A C C A U U C U A U A G C A C A G C e G A U G G G A C c U G A U U G G U G G A A A G U U C U A A A G C A C A G G U G G A C U G U U C UC A A A G C A C A n U U G CC G U G G U G G G C U G U U U C A A C A G C A C e C u A U AC A C GC U G G G AA A C U G U U G AC A A G C A q C e U U U U GC U G U U A A A C U G AA GA G AC A A G C C A C U A UA G U G G U A A C U G A A A G AC A A G S U G U C A G A GC U G G UA U U A A C UC U A A A A A A d G C U G U G U G A G U G U U U A A G C A A A U A A A A A n U G a A A CC U U C C A G U UC A U U U A U A U A A G C AC A U U C A C UC G U C G U U A U U U U C U A U A AA G rtS A A A A G U A C U G A U C A U A U U C U U C U A U A A G e G U A CC U A A C G A U U CC A U A U U U U C U A U A A s A A G G U n A G U U A U U A C U A U U A C G A G C C C U G U U C A U A U C C U U C U A A C A U A G U G U U C U U e A A C G A C U C C A U U A S U A A A A U A A C G A G U U A A U U C A C G U U C A A C C A A U U A U C C C C A C C A U C U G U U U C U 88 98 71 32 82 92 03 13 23 33 43 53 44 46 56 66 76 86 96 67 77 8 9 0 1 2 3 9 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 70 70 8 8 8 8 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 01 01 0 0 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000266_0001
' 3 A A A A A A A A A A A A A A A A A A A A A A A A A A AC A ot U A C C U G A U A G G A U A U A C U U C G U A U U U U U UC ' U G U C U U C G U G AA GA G A G G UA A U A C U U UC G U A U UA U U U U U 5 U U C U G U A U A C U U G U U A U A A G A A U C A U U U C G U C U G A G A U U C U G U ec A G C G U C U C G AA G G A U A U A U U C U G U A U U n A U G G U U G C U U C A G G A U A U C A U U C U G U A U e A U A A G GC G U U UC G A A G G A U A C U U C U G U A u U AC U U A G C G U UC G A A G G A U U A C U U C U G U qeS A U A U G C A A U A G C U G A AA G G A A U A CA U U C U G U A A U A G GC U U C G U U U G C G A G G U A A A A U U C A U A U U C C U UC dn G U U A A U A G C U A U G G U A A U A C A G A U G GC U U UC G A G A U UA A C U U a A U G G G U A A U A G G U U U G A GA G A UA U A CA U rtS G G G G U A A C U A G U U C UC G A G G G UA A U CA U UA U G G G G U A A GA C G U U U UC G AA G U A UA es C U U U G C G U A G G A U U n U U C U U G G G U A A C U G A A U A C G G G U A UA A GA G G U U A C C A GA G G A e U G G UC U U G U U G G U S G U C A C G U G G G U A A C U C G A G A A G U C U U G G G U A A U A U G A G G C U U U C U G A A G A G G 69 79 99 00 10 20 30 40 50 60 70 80 90 01 11 21 31 41 51 61 71 8 9 0 1 2 3 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 11 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 11 1 1 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000267_0001
' 3 A A o C A t A A A A A A A A A A A AC A A A A A A A A A A A A A U C G A G U U G A U U G U G UC U AC C U A G G A A A U A ' C 5 U A G A G G U G A U U A U A U C U A G U C C U U G G A A A U A A A G U G A A U U G C U AC C A U G G A A A U AC U U G U A G U G U C A U G U U U AC U A U G G A A ec U U A A C A U U A G U U A U A C U U U U C U A U G G A n U UC U C UA U U U A G A U U A AC UA AC C U A U G G e U U U C U C A U U U A G U A U U U C UA U U A C U A U G u A UA U U C U A A U U U U U G A U U U C U C A A C U A U qe U U U U C C A A U U G U U G A C U U C U U C AC C U A S G U U U U A C A A U A AC G U G G C U U U A U U U A C U U G U A A U U U U C AC A U U A G U U G C U C UA U C A C dn C U a U C U A U U G U A A AC U U A G A U G C U C U U U C AC rt U U U A U A G U A U UC U U A U A U G U U AC U U U U S C G U A G A G U U A G U U U U U A U C A A U U C U U U C U G G U A G A GA A U A U U A U U A G C U U AC U es U A C U U C U n C G U A G G A U A G A CA A AC AA UA G A U U U G UC U U AC UA A U A G U U A C G A A U G C C e A U U U U C A U A U U U U S U A U C C C A U A U G A U A G C U A U C U U U C U A C U A U G A U A G U C A G A G U G U U U A U G U C U 42 52 82 92 03 13 04 14 24 34 44 74 84 05 15 25 95 06 16 26 36 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 61 61 6 6 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 11 1 1 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000268_0001
' 3 A A A A A A A A A A A A A A ot U U G A A U U G C G G U A AC A U A A A A A A A A A A A A U C C U U A U G U U A A U G C ' A U U G A A G U G C U G U G U A C C C C G U U A A U G 5 U G C U G A C U U G G U G U A UC C A U C G U U A A U e A U A C c A A U A UC G G U U U C G U A U U G U A U U C U C G U U A A C U G U U G C GC G G U A A A C U C G U U A n A U U U U A U A G U U G G C U U G C CC U A C U C G U U e G A G U u G A U U U G A G U U G U G C G U U U A U A C U C G U q A U G G G G G A U U U U G C G C U CC A U A C U C G e U G A U U U A U G G G U U U G C U C U CC A U A C U C S A U A A G G A U G A G U U U U G C A U U CC AC U A C U d U G A U U UA G U A U G A GA G U U U G U C C AC U A C n C U AA AA A U C U A G G G A U U U G A UC U U C A U A a AC A A A U A G C U U G G G G U A U U A UC U U CC A U rt U UC G A G A A U A G C A U G A A G A G G U A C CC A S G U A G G A C CA A G U A U G G G A U C U G U A UC U C es U C U n U A U G G G C A A G A C C U A U G G C U A U G G U G G U U C G U G U A C U U AC U U A U G C A C U G U C G U A C C A U A U G C G A G UC A A C U G GC U U G U A U e S U U A U C U A G A U C U C A A C G A G U C U U A U C U U G G C G U G U C A 07 37 77 87 97 08 10 04 14 24 34 44 54 64 74 84 94 25 65 85 95 0 1 2 3 4 5 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 62 62 6 6 6 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 21 21 2 2 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000269_0001
' 3 A AC A A A A A A A A A A A A A A A A A A A A A A A AA A ot UC U C A U C U U A U U A A A U A A CC C A U A U G A ' UC C U C AC AC C U U A U U A A A U A C C A U A U U G 5 G G UC C U C A C U U A U U A A A U AC C C C A U U A G ec UA U G U C U C U C U CC AC C U U A U U A A A G AC C C C A C A G C U A C A C U U A U U A A A A C C C G CA A n A AA U G C C U U CC A C U U A U U A G G C A C C U C C eu U A U G G U AC A U CC A C U U A U U A A G C A C U C U q U U A A U U C U C A C A C U U A U U G A G C AC C G C e G U U A A S C G U U A A U C U C U C C A C U U A A C A A G A G C A U U C d U C G U U U C U AC U C C A C U U C U A G GA A U G U G C U C U AC U C C A C U U AC U A GA U C n C U C G U a A C U G C U C AC UA C C A C A U A U G U A A G C rt U A UC C G U U C G A U G C U UC C U C C A A A C AC U C U C S A U A C U C A e C U A U G C U A A U G U C C U AC U C C U A U AC UA C U U C A U C A U AA U C C UC G s C AC U A C U n C A U A C U U A A U G C U UC C A U C U A A A A U A U A A U C U G U C A U A A G C A A A A G A A e U C A U G U S U C U U C C A C U C U U A U U C U C A U G C G U U A A U G C C U A A A U G U C U U C U U A U A A A U A A A U G U A U A 66 76 86 96 07 17 47 57 67 77 87 97 08 18 28 38 48 58 79 89 99 0 1 2 2 0 7 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 03 03 0 1 2 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 31 31 3 3 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000270_0001
' 3 A A A A A A A A A A A A A A A A A A A A A U A A AC A A A ot U U U U A A A G U C U U G A U A U G G U A U U U U A A A G U U U A G A U G U G U C G U U UC G A U U G G U UC G ' G A U U U U A A A G A U A A G A A G U GA U A GA G G U 5 ec G G A U U U U A A A A A G C A G A A G C U C G G G A U U U U A A U G G U A G G n A C A C A C A G A A A G A U A U G e C G G G A u A G G G U U U U A U A U U U U U C A G A C C G A U G UC G G A U A U U U G A A C G UC G U U G G A U q U e C C A U C G G A G A U U U G G G G A U U AA G A U U G U A C A U U A G G A U A A A U U A U A U S C G C G U A G G A C C d C UC C U AC G G G A U A G G G A A A A A A U A A U U A A A U U A C U A G G n G C C U AA A A A A A G G U C U A G a U C C U C A G G G U U U AA A A A G U U U U C U A rt C G C C U C A G G U U A U U A U U A A G A U U U U C U S C U G C U C C C U C A G C U U U G C G C C C U C A A U A U A U A C G A U G G C G U U U C A G A U G U G G U U U es G U C U n G C G C CC C U C G A A G A AC G A U G G G G G U U G U C U G CC C U G G G GA A GA A AC G G G G G U e A G U CC U G G CC C G U A G G A A AA U UA U U G G G S U G A G G U C C U C U G C C G A G U G A A G A U G C A G G A G G A G U G G 86 96 07 17 27 37 47 57 67 77 18 28 60 51 61 71 72 82 92 24 44 9 1 2 3 4 5 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 44 74 7 7 7 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 41 41 4 4 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000271_0001
' 3 A A A A A A A A A A A A A A A A U A A A A A A A A A A A A o A AA A U U G G C A U U U C A A G U C C U A U G U G U U t G A AA A U U U A C A U U U G U A A G C C U A U G U U U '5 U G A A A U U C A C A U A G A C G U G C C U A U G A U C U G AA A A U A C A C A C U G U U G U G C G C U A U G A ec U C A A A AC AA C A C U U G A G A G U C CC U A U G n G U UC G A AA C C A C A A U C A A A G U G G CC U G U e G G U UC G U G G AC A C U A U U A A A A G U G CC A G u A G U G U U U G AC A A U U A G A A AA AA G U U G A A q U A G U C UC U U U U A U U U U U U A A A A GA G U G A eS A U GA G U U C U G C A U A A G U U U A AA A A G U G G A G U G G G AC UA U U G A C U U U U U U U A A A A G U dn G G A A G G U U C U U U A A G U A A U U U U A A A U G a A G G U A A A U U U C U U U A U A U U U U U U U A A A U rt U A G G UA U A U U AC U C U C A CC G U U U U A U A S C U A A A UC U U U AC U G GA U A G U U U U U U CC U es U C n U U U G C A G G G A A G C U U U A A C G C A G U U U U U G CC U G U G C AC AA U U U U C AC G U U U U U G e U U U C A A G U U S G U U U U C U U C U U G C A A U U G U C G G G A A A G AC U AC G U U U A A C A A U A C U A U A C G A G U G A G 67 77 87 97 08 18 09 69 79 89 99 00 11 12 42 94 26 36 46 56 66 7 8 9 0 7 8 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 65 65 6 7 7 7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 51 51 5 5 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000272_0001
' 3 A A A A A o U U A A A A A A A A A A A A A A A A A A A A A A A A AC t U U U A A G C A U G U G A A G G U U A CA UC A A U U U U A A G U A U G U G A A G G U U U A U A A U U U ' U U U U U G A C U A U G U G A A U G U C U A U A C A U U 5 U U U ec A U U U U A G C C U A U G U G A U U G U U U A UC A A UA n G A U U U A A A G C C U A U G U G C U U G U A UC A A e U G A U U A AA A G C C U A U G U U C U U G U U U A UC U u G U G A U A A G A G C C U A U G U U C U U G G U U A C q A e G U G A U A AA G A G C C U A U G U U C UC U U G U A G U U A A G A G C C U A U G U U S A U U U C U U G U G A A G U A d U G A G U U U A A AA GA A G GA C C U UC U G U U U U G U U C U U G U A A G U U A A A A A G G A C C G C U U G U U C U U G na G U G A A U U U A A A A G C U U C U rt U G U G A A U U A A A A G A G U G C U U G G U U UC U S A U G U G U U U U A A AA A G A UC U G C UC U U G U C U G A U U U U A A AA A G UC U G G U U C U U U e U A s U G U G U A A U U U G U C G U U U U A A n C U A U U G A A U G C G U G CC U A A A U A U U U U A A A A UA C UC G UC U G A G G UA U U U U A A U A U UC U U G UC U e S U G C U A U A A U U U U C C U G A U G A U U U U U A G U A A U C U U G C U 97 08 18 28 38 88 98 29 39 49 59 69 79 89 99 00 10 20 30 40 50 6 7 8 9 0 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 06 06 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 61 61 6 6 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000273_0001
' 3 A A A A A A A A A A A A o U A A A A C U A A A A A A A A A A t A U A U G A A U A G U U U G U A U U G C A G U U A C U C U ' A U A 5 G U A AA U G A U U G U U A U G UC U U U A G U UC U U C A G A C U G U A U UA U G UC C A U U A A G U e C U U c A G A GA A U G A U G C C U G U U A A U U C C U UC A A U G C U A A G G U A G A U U A G C G U U U A U A n A U A A A G A A U U C C A G A A A U UA A A G A A U G U e G C A u A C C A A G A U U U C U U U A A A U A C A C U U U A G q U C A C A A G A U U U A G U A A A U U G C G C C G A e A UC A A C A A U A U U U G G U A A A U U U C A U U G S U U A U C A A A C A A U A U A G G G U A A UC C A C A G C U d A G U A A A C G A U A A U G G G U A A U A U U A A C n U U U A U A A A U G A UA A U U G G G U A C A A U U A A a A A G A A U A A A U G U G U U G G G C AC G A U GA U A rt UA G C AC A A U A A A U G G G U U G G U A A G CC U S e C A A A A U G A A U G U U A A U G U U G U G G A U U CC s G UA U UC C A A A A G A AA G A U G U U G U G A U UC C n U A G C UC C A A C A A G U U A U G U G U U G A A UC e C U U A C A A G U U C C G S U G G C C A U U G C C U C U A A C U U A G U U A G A A A C A A G C A U C U U U A U U A A U C U C G U U G 64 66 48 19 29 39 49 59 20 30 40 50 71 13 23 33 43 53 63 15 85 5 7 7 4 1 2 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 8 8 68 68 1 2 3 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 91 91 9 9 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 1 5 1 1 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000274_0001
' 3 A A A A A A AC A AA A A A A A A A A A A A A A A A A A A ot U A U A U U AC A UA A A U U U A G G A U U U A U G U U ' U U A U A U U U C A U G A U A U A U U A A U A A U G U 5 C U U A U U U U U C U G G A U A U A A G U C AC A A U G e U C U U A A U c G U C U U U A U U U A A G G U U A CA A U G A U AC A A U A U U G C A G A U U A A A U U A A C A A n A G U C U A UA U A U G A C A G A A G A G U G U U AC A eu U A G U C U A U A A A A C G G U A A G C UA UC G A G C q G U A G U U U C U A U C U A A A e A G U A G U S G A G C C G A U C A U G UC U U U U A A U U A A A U A A U U U U G U C G A U A U A G U G U C U U C U U UC U U A CA UA U U A C G A G U A G U G UC U d U G U C U C U A A U U A U C n C U G A G A G a A C U G A U A G U C UC A UC C U U G U C A G U A G G rt A A C U G G U A G U U A U C G A A A AC C UC U U UA U S U A A C U A G U A G UC U U CA U UC AC U C A U A UC A A G U A A C U A U U C G C A U U A UC U es CC U A C U A C G A A U G A G U U UC U U A n U C U U C A A G G U A C G A U A U U C U C C A G A G U A G A U A U A C e S C C U C A U C U C C U C A U A C G A U U CC U U U U A U C U G U C U U C C U U U C G UA UC U GA A U A U A A C A A C U A U A U 33 43 53 63 73 83 93 04 14 24 52 63 73 83 93 04 54 15 66 37 60 2 2 3 4 5 6 9 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 1 11 21 2 2 2 2 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 12 12 1 1 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 2 5 1 2 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000275_0001
' 3 A A A A A A A A U A A A o C A A A A A A A A A A A A A A A A t A U U A U G U U A G U U U U U UC U U A CA U C C U U U A C U U U A A U U U G U U U U CA U C A U C A U AC G U '5 U U U U U G G U G U U C U U U C U A U U U U U G G U G e G U U U A A U c U G A U U U U G U G U U C U U C U U A A U U A U A U UC A U U AC G U G U U C UC U G A U A U C C n A U U G U A A U AC G U G U U U U U C U A U C A G U A U e A A A U G U U U A U AC G U G U U UC U C A A A U U A U u A A U A U U A A U A U AC G U G U U U C A G C G U U G A q C AC U A A A G U U G U A U AC G U G U U A U U U A U G eS U G U G C AA U U A U A A G U G U U U C A C A U AC AC dn U G U U C A G U G GA U C G U A A G U G G C A C U U G G A a C U A G U AA U G A rt G C A A U G C A U C G A A G G U UA C AC G U C U G U U A A U A U AC G U UC U A U A C C G S U G C C U A A U C A G U A UA U AC G A C C C C U U G U G UC G AC AA U U C G A G U A AC U C G A A G U e A U s U G C A G A G U U U U C A U A U n U U U A G G U U C A U A U C A U U U UC U U U G U U C G A G U A A A A A U U A UA e C U A G G G A G U U AC G GA U U UC U A AC GA S C A U C U U A G G G U A G U U U C U U A U G U U C A G A G U U U G G U G U 72 82 92 13 23 43 53 63 53 63 73 83 93 04 14 24 34 44 54 45 75 2 5 6 3 1 8 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 33 33 3 7 8 8 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 32 32 3 3 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 2 5 1 2 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000276_0001
' 3 A A A A A A A o U A A A A A A A A AC A A A A A A A A A A A A t C G U G U U U U G C U U U U U U C U U C U U A A U A U U G ' U C G U G A G U G C U U U U U A U U C U U A A A A U U 5 U U U U U A U G U G A C U A U A A U U C U U A G C C A ec A U C G U U UA G C A UC U G A A U U C U U U AA U CC n G UA U U G U U G U U G U G C A G A A U U C U G A A U e A U C U G U U G U A U u C G A U U G U A G A A U U C A A G C U A A U C UC G U U G U C A U C U A G A A U U U U U U q A C G e U A A U U C A U U UC G U U G U U U A U C U A G A A U G U A S G U A G A U U UC G U U G U G U A U C U A G A A U A U A A A U G U U A U G C A U C U A G A G A A U A d G G U C G n A G UA U C U G U A A U U C C A U C U A G U C U a U G G A C A U U C U G G U U AC C A U C U A GA U G U rt UA U G U A C GA A U U C U A G G U U G U A C G G AC C A U C U G G U U A A U U C U UC U U U G A C A U C A U S A A e U G G U A C G A U U C U C A C U C C A A U U G C UA s U A n G A G G U A AC G A U U U AC U G U U C C A UC A AC C U A U U G G U A AC G A A U G AC U G U AC C A G G e U G U A G C A U A U G A G U U G G U A A G G C G U C G U S U U A U C A A G A U A U G A A U U G G U A C C A G U G G C A U G U U U U U 72 82 92 03 13 23 33 43 53 63 73 83 04 14 24 87 97 08 18 28 38 4 5 3 7 1 9 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 84 84 1 1 6 9 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 52 52 5 5 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 2 5 1 2 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000277_0001
' 3 A A ot G AA A A A A AC A A A A A A A A A A A A A A A AA U U A A U U A U U C A A A U U C C U AC A U U U A C G A A ' A A A U U U U A U CC A U U U C C C A C C U U G U U A 5 UA A AA A A U G U U A U A U U U C G AC G A C U U U A A ec G A n U G A A A A A A U U A AA A A A A A C U C C CC A U U U U A U A A C U C A U A U U C U U U U U U C A A A G A C e A U G A A A G U A U U C C C A U U UA A A C A A U G G u C C A U A A A G C C A U C U A U U A A AA CA A U U G q C U C G A A eS U A U A A A A U C U C U A U U U UC G A G U U A C A C C C U A A A A A A A U U A UA A U U A U A A U U U A U UC G U A d U G C G U C A A U A U C C A C C U C G C U G U A G U UC A A G U n U G U A U C C A A U C A G U A a A A rt A C G U A G A G U A U U A C A A C A C U G G C G G A A U U U A U U A A A A A A U A C UC U A U U G A U U G S A G C U U G AC G e A U U G U A A C A A C U A C U AC U U U C C UC G A C C A A A A A C U U U A U U AA A U UA s U U U G C A n U G U U G A C A A A A A C A A A C U G A G U C U G U A C U A A G A A A AA C A A A U U A A G U C A G e S U G G U U G A U G G U U U A C A U C A A A C A A A C A A A A A A A C A A A U A A G A A A A C A A U A G G A C A A A A G A A A U A G 30 73 83 93 04 14 00 01 34 94 86 07 17 27 47 57 67 88 98 40 60 7 8 5 7 4 8 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 8 9 9 09 09 1 1 4 6 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 92 92 9 9 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 2 5 1 2 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000278_0001
' 3 A A t A A A A A AC A A A A A A A A A A A A A A A A A A A A o A A AA A ' U A C UA A U A A U C U U C C U U U C A C U A C U U A A U A AC U U C C U U A C G G C U A C U U A U 5 U C U A U A A UA A U U C C U U A AA A G C U A C U G G ec UC A A U U C C U A U U C C A U A A A U U C C U U C A A G C U A U A U A U A U C A U U C A U A C A A G C U G U A n C U U U U AC A A U A U C A U U C A U A C A A G C U U A e U u A A G C U U C A A AA U C A U U C U U A C A A G A C A q U C U U U C U U C A A A e A U A G U C U U C A U A UA C A A U A U U A C A A A A G A U C C A C A U U A C A A U A S C A A G U G U C U U C A UA A U A C U C A U U A C G C U d U C U A U A U G U C U U C A A A C A A U C A U U A A G U n A A U A U G A U G U C U U C A A A C C A U C A U U U A C a A C U G A U G U C U U C A UA A A A C A U C A U U AC A rt U A G U U G A U G U C U U C A A A C A C A U C A C A U S A A AC C U G A U G U C U U C A AC A C A C A U C A U C e A s C AC G GA U U G A U G U C U U C A A A C A C A U UC G U A G C GA U U U G A U G U C U U C A A A C A C A U A n C G C G G U U G A U G U C U U C A C C A A A C A C A C G AC A e S U C U U C G G A C A G U U G A U G U A U A A A A A C A A C A A U C A 95 06 36 56 66 76 86 96 07 17 27 37 47 57 67 20 30 40 50 60 70 8 9 0 1 6 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 01 01 1 1 1 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 13 13 1 1 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 3 5 1 3 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000279_0001
' 3 A A A A A A A AC AC A A A A A A A A A A A A A A A A A A ot A U A A U G GA G C U U C U U U U U A A A A U C C A U U ' U U A A U U A G CC U G C U U A U A A U U A U C A A U 5 G A U A A A U UC A G CC U G C U U A U A U A U A U A G U e G c U U A A C C U A A A C U C U A A U C U G U U G C U G A U C C A U A A U C A CC U U G U A U A U C C A U A A A n U C C A AA G C A U C G CC U U C G A U C G C C A U A C e U U A C A A U C A U UC A G C U G A U A A U G C C U C C u G U A A C UC U U C A U U A C C U C U U A C U G C A U C qe U U U A U S A A A U U UA U U U C A C U GA C U U A U G C C U G G U U U U U U C U C U G C C G A A C C C U U A A d A U A A A n A C A A U C U U U U A U C C U A G C U C C C C CC AA a G A U A U U U U U U U U C A U UC GA C G A G U U C C A G A rt A U C U U U U U U U C A U A A G A G U U C U U U S U A U C A U U U C U U U U U C A U U C A UC U G U U U G A U C U UC U U U U U U C C G U C A C U G U U U G e U C U U s C A C U G U U U C U U U U U U UA A C U U A C U G U A U n A A C G U C U U U U C U UC C U C AC C U C A A e A G G G U U U U S U C G U C U U U C C U U C U U U U U U A U A U C U U U U U U U U C C C G G A A G C CA AA U G U A A C A G C C G U 52 10 11 21 98 02 43 53 63 73 83 93 04 14 24 44 47 40 50 28 22 3 4 5 6 9 8 1 2 3 3 3 4 4 4 4 4 4 4 4 4 4 4 5 6 6 6 7 27 27 2 2 8 0 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 73 73 7 8 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 3 5 1 3 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000280_0001
' 3 A A A A A A o C A A A A A A A A A A A A A A A A A A A A A A t U U A U C U A UC U AA A A U A A G G U G A G A A U U A U U A U C U A C A A A U A A A G U G A A U A U G A C A A '5 U U C U A U U U U A C A U C U A UC A A A A A U C G A G U G A A A G G G U G A A A U A G A G U A G G G U G U A G ec C U U U U C U A UC A A C C A G A G A G A U A U U U A n A G U U U U C U A U A A A U C A G A G U U A A A AA A U e C G G U U U C U CA UC A C C U C A G A A G A U A G U A u C G G G G U U U C U U A C U A C U C A G A U G U U A G U q C U G G G G G U U U C U A C C A C U C U U U A A U U A G eS U U U U G G G U U U C U A U C A C U A U G U A A A G A A U U U G G G U U U C U C U C A C AC A A G AA A A A U G dn A C U U U G G G U U U U A CA UC C A U A G U U G G A a AA U C U U C U U G G G U U U U A U C G A A U A A A U G rt U U UC U U U G G G U A U U U CA U A C A G U G G G U S UA U U U U C U U U G G G A U U G U A A UA A A A A U G es G U U U U U C U U U G G U AA U U A U G G G UC G G CA U n U C U U U U C U G A A UA U U G U A A U A G C U A CA e A A C U U U U U A U U U A S G C C A C C U U C U U A U C U U U U U C U U A A C A U G G G A A A A U A U A A U A G A G U C G G U U G G 90 02 12 22 32 42 52 62 72 82 92 03 13 54 64 74 84 94 36 48 59 9 4 5 5 3 4 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 98 29 2 3 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 93 93 9 9 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 3 5 1 3 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000281_0001
' 3 A A A A AA A A A G A A A A A A A A A A A A A A A A A A A ot U U C U U G U C A C U G C C U A U C C A C A A A C U U U ' U U UC U A G U G UC U U U C A U C CC U A A A A C U C 5 A A U U A U U UC U A G U U A U G U U U U U G U C G U G U U G U U U CC A A A C U ec G A A U U C U A U G C C U C A A A U A U U G U C C A A U C U U C U G A C U C C A A C n A G U U A UC U UC A AC U U C A CC U A U G U C C A A e U A GA A U U U U U U U U U U C A A CC A U U G U C C A u A U qe U A U G AA U U C UC U C C U G U U A A C C CC U G U C A S G U A A G AA U U U U U U A U U U U U A C A A CC U G U C U A A U U G A C AC A A U U U U A A A C U G C A G U A U GA A A U U U U G U A A G U U G G U A CA C U U dn G A GA U A G A A U CA U G A U C U U U U U A CA C G a A G U A GA A A U A G A U A A A U GC G C A U U U A C U rt G A G G U A A A U GA U U C A U G U C CA A G U AA C S U G G U A U G G G G A A A A A G A C U G U A C C U U CA e G U s U G U A G G n C U G G A A U U U A U A G C G U U U U G U A U GC U U U A U G G G U A A G A U G U U U C G U U C G U GC U U U A U U G G G U AC AC A U U U A U G U G U e S A A C A U G G G U C G U C G U G A G A G U U U U G U U A G G C U U G U C U 55 65 75 85 95 06 16 26 36 46 60 56 50 90 13 96 39 49 59 69 79 8 9 0 1 2 3 9 9 9 9 9 9 9 9 9 9 0 0 1 1 1 1 1 1 1 1 1 91 91 0 0 0 0 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 24 24 2 2 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 4 5 1 4 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000282_0001
' 3 A A o A A t A C A AC A A A A A A A A A A A A A A A A A A A A A A A A U G A A C U U C U U A C A A G A U U C G U U U ' U A C A C A A U G A A C U U C U U A C A A G A U 5 C U A C A C A A U G A A C U U C U A A C A C UC G C A G A U U G ec U C U U UC A C A C A n C U A C A C A A U G A A C U U C U A A A U G A A C U U U U A A AC A G U C A A A AC A G U G e A UC U C U A C C U A AC C A A U G A A C G A A U C C U U U C U U A A AC A U A u U U A C A A U A U A A A G A q AA A C U U C UC A C A C A A U G A U U C UC U A U A U eS C A A C U U C A U UC A C A C A A U G A C U U UC U U U A A C A A C U U A C AC C A A U G AA C U U U C U U U U dn U C A A A C UC U C U A A AC C A A U A C UC U C U U G a G U CC A A A A U U C U A AC CA AC A G A A A UC U U rt U CC AC A A C U U C UC A C A A U G A A A UC U A U C S C G U A C U U C C A AA C U U UC A C C A U G A A C G U U C G U C C A A C U U U A U C U A C A A U G A A A A C es A n A C U G A C U C C A A C U C U A C U G G U C AC A A C U C U A A A A U G G A AC G U A CC U G U C A A A U A C U C U C C A A U G A A U A UC U C A A U C C A A C C A U A C A A A e S U U A A C C U C U G U C C A C A A A U C U U A A A U A U C A G A 40 50 60 70 80 90 01 11 21 31 41 51 61 71 81 91 02 12 22 32 42 5 6 7 7 9 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 22 22 2 3 3 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 24 24 2 2 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 4 5 1 4 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000283_0001
' 3 A A A A A A A A A A A A G AC A A A A AA A A A A A A A A ot A U U A G U U A A U G A G G C G A U A U C U A G C AA A U ' C U U U U G U U A A U 5 U A U U U U G U U A A U G G AC GA U U A U U A A C C U U C U A U U U U G U U A A U G G C U U G C U U U C G U ec C U A A U G G U U G U G U U U G U A A G U U A U U U G U U A A C U U G U A U U A A U U U n C A G C U A U U U G U U U A A U U UC U A U C U A C G C e U G A U U G U A U U U G G U U A A U U U G U G C A A A C u U A GA A G U A U U U U U U G U U A U A A U U G A A C C qe U U U A U G A G U A U U U G U U U U U A C U U U G U U S G A U A G A G U A UA U U U G U U U U A A U A U A U AA d G UC U U A G A G U A A U U U G U G U UC U U U U UC C na A A UC A A U A G A G U U A U U U G A U A C rt G C A G UC A U A G A G U A U U A A A G U A U U U G C U A A A UC A S A G C U A U A G G A C C A U A G A A U A U A G G U A U A G A G UC C U U U U A G A G U A G U A A C U U C C A G es U U G G G C C n A U U A G G A U A U A C C G A A G U U A A A G G CC A U C G A U A U U A G A G G GA CA A A A U A e U S G U U G U G G C C U A A U A U A C U U G G A C C A A U C U U A A U G A C C G U A U A U U A G G G A G A G A A A A U C A A U C A U 06 99 00 10 20 30 40 50 60 70 80 90 01 11 21 31 82 92 25 98 00 3 6 1 2 3 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 04 25 6 6 6 7 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 54 54 5 5 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 4 5 1 4 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000284_0001
' 3 A A A A A A o A A A A A A A A A A A A A A A A A A A U U A A t U A A A A C G C U C U U U C A G U U G U U U U C U U U CC ' U U A A A C A G C U C U U U C A G U U G A U A A 5 U U U A A G C A U C U C U U U C A G U U C U U U A A C U A U ec UA U U U A U U U U U C C C G C U U C U C U U U C A G U U U U C U C U U U C A U CA U U A G U A n C G A U G U UA e U u C A U U U A U G U A U U A C U U U U U C U C U U U A U G C U U U U C U C U U U C A A C U G U G A UC A C U U G q C C U A U G A C A C U U U U C U C e C C C U A A A A U A C U U U U C U U U U U G C G A C S U C C UC A U U U G A UC A C C U C CC U U G A C A A G AC U A CA UC U U U U U U U U C U C G A U G U G A dn A A U U U C U A G U U G U G a C A U C rt A AC A C A U A G C A U A C U U U U C U A A A U G U A U C U A U A G C AC U A C U U U U U G U U G A U G S A U AA C A A A U A A A C e C A A U A C AC G A U A C C A G C A U A UC U U U G G A G A A U U U U U U C A G UA s G U A A A G C C G U A A A A U G G CC AC G C A U CA UC C U C A A A A U G C AC G C AC U A UC G U A U U C G ne A G C A C U A G A U A G A S U G U A A G G U U C A A C G C C A C G U U U C A A A C A U C U G C C A C G A G A C U A U U A C U U U U U 37 47 57 67 77 18 28 38 39 49 59 69 79 89 99 00 10 20 30 40 71 8 6 7 8 9 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 16 66 7 7 7 8 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 64 64 6 6 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 4 5 1 4 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000285_0001
' 3 A A A A A A C A A A A U A A A A A A A A A A A A A A A A A ot U G A G UA A U G G C G A U U G C A A U G G C U C C C U ' C 5 C U U C C U C U A U U G U C G G C U C U G C A A U G G C U C C U U G UA AC U U G C A A C U G C A U U G G C U C C U U C C U G A U U G C A A A C U G C A U U G G C U G ec U U C C U U C AC U U A C C A A C U G A A U U G G C G n U A A U U U C C U AC U U G C C A A C U C A A U U G G U e A U U U C G A UA U AC A U U C C A A C G C A A U U G U u U U A U U U UC G G A U U C A U C C A A U G C A A U U A qe G G U A U U C U G A U A AA A U C C A C U G C A A U U S UC G U A U U CC UC U G C A A A A U C C A C U G C A A U dn A UC U G U A U U C C C U C C AA A A U C A A C U G C A C G A C U G U U U C C A C UC A A A U C A A C U G C U a G A C U U G A U U C U U G UC A A AA A C C A A C U G A rt U U G A C U U U U U U A G G UC A A A U C C A A C U C S G A U G U G A C A U U A A G G G U A C A A U C C A A C G e U s A U G U G A G U A U U A U G A C G G G C U A A U C C A A A A n G A U G U G UC G U A A G C G C A AA A U C CC A G e A G U G U G G A A A U C S A C G U A G U G C U G C G G A C A A G U A U G G C A U C U G G G G U G G C G G U C U A A A A A A U A U G 18 28 38 48 58 68 78 88 98 09 19 62 37 98 09 19 29 39 49 59 69 7 8 9 0 1 3 6 6 6 6 6 6 6 6 6 6 6 8 8 8 8 8 8 8 8 8 8 98 98 9 0 0 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 84 94 9 9 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 4 5 1 4 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000286_0001
' 3 A A A ot A U A U A A A A A A G AA U U A A A A A A A A A U U U A G A A G A C A A A A A A A A A A A A U U A G A A U A G A U U G U U U C C A U G A U U G U U U C C A A A CC C '5 A U A G A A U C U U A G A U C U U G A U U G U U A A CC e U A G A G A A c U U A C U U A G U U G A G A A A C U U A G U A U U G A U U G U C A A U U A A U U G A U U U U C A n C e G UC A U G A G G A A C U U U A A A U U G A U G U U C u G G A A U G A A G A A C U UA U G A A A A U U G A U U U U qe U G A A A U G A A G A A C G U A G A A AA U U G U U U U U A A A A U A A A G A A A G A GA G A A U U A U U U S U G A A A A C A A A G A U A A A A A G A G A A U G A U U d A U A G A A A AA CA AC AA A G U U A G G A A A A A U n U A C A G A AA A A C A A AA U G A AA A G G A U U A A a U U U C A G G rt C U U U C A G A A AC AA A U A G A A A GA A U U U A S U C C U U C GA U G A A C G G A G C U U A G A G G A U U U U G A A G A G A G A G A G A A A A G U U e A U U C s A U U A U A G A G G A C U n G C AC U C UC G A U U G A U U G G U A G A A U U G A G G A G G U A G A G U G G e A A U UC A G C U G A A S A A G A U C A A C U A U A G C G A A U U G A U C A A C G A G U A U G U U C C U C U G G A G G A G A G U U 64 74 19 29 39 49 59 92 24 34 44 54 64 18 28 79 89 99 00 10 20 3 4 6 7 8 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 0 1 1 1 01 01 0 2 2 2 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 15 15 1 1 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 5 5 1 5 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000287_0001
' 3 A A A A A A A A A A A A t G U A A A A A A A A A A A A A A A o U C U A U G C AC U G G U U A A G C G A G G C A A A U G ' U U C U A U G A U U A U C U G A U A CC G C G AC A U 5 A U U C U A U G C A U U A A A A U A A U U CC GC U A A e C A U U C U A U G C A c U C A U U C U A U G C A A A A A A C A C U G A C U UC AC A G U U U C A G A C C A C GC C n U U C A U U C U e U U U C A U U C A U U A U A AA C A A U A U U U U C U C GC u A U A U A CA A A A UC U U G U U C q U U U U C A U U C U e G U U U U C A C U C UC U G U A A A A U U CC U U S A C U U C C U U U C A U C U A U C U U C U U d C G U U U U A A G U U U U C U U U G G U U U C A C U U U U U U C U U AC U n A C A G U U U A U U U AC U G U A G U G U U U U U C a U A C A r G U U U U C A G A U U G U G A A G U U U U C t A U A C A G U U U U C U U G A U U C A U UC A G UC U U S C e C A U A C A G U U U U G U U A U U U U A U U A G C U A G U U U U U A A U U G A A U U C U U A G s U CC AC U A C A A U A C GA U G U U U AA A UC A U U C n U C AA A U U U G C U A G U C C A C U A A G C A A U C e C U C A U S U G C G U C C A C U A A A U C G A A C G G A G A A U C A G U C U U U A U C A A U A U UC A U U C G U A A A A U C U A U U 86 96 07 17 27 37 47 57 67 77 87 82 53 63 83 14 24 54 69 87 88 9 0 1 3 4 5 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 83 93 9 9 9 9 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 35 35 3 3 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 5 5 1 5 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
Figure imgf000288_0001
' 3 A A ot C A A A A A A A U A G U C U U A A A A A A A A A A A A A A A AA A A A U A A C U U C A U U U A A A C A U A A A A ' A G CA UC A U G AA UC G U A U A UC A A A U A G C U UC A A A G U G U U A A 5 A G U U A U U U C A G G U A e U G A C U A c A U G A C C A A U CC A G U A C A C U A A A A U U A U C U A C U U A U C U C A A C A G A G U U A C U n AC A U G A AC U U G U U A U A A A A G U A A U U U U A e G AC A U G U U U G U C A U C U C G A U G A A U U U C U uq C AC A U A U U G A A G G U A A U G U U G A UC U C A G e C GC A A U A G A A U G A U U U U U C C U A A C U U U S U C GC C A U A U U C A G U A C U U C U U A G G A C A C d U U C GC C A n U U U C GC G U G U U A U C U C C A C C C U U U G A G A G A A U U A U A U U G C A C C G A A U U A a U U U U C AA UC U U AC U U U A U G A A A U A A A C A rt C G U U U U G A U G U U G C C A U G A UC C U A G G U S U C U U U A A A U U A UC U UC A C A U G A A G U U A A es U U C U U n G G G G G U U A A GC A A G A C U U A U G C U AC U U C U A G UC G G G G A A A CC U G C U A U G C G e A S U G U U C G G U UC G A G U A U A A A G C U A U U A G G A G U U U C G U C U A C G A A U U G U A C A A U U C U G G C G U C A A G 69 79 89 99 00 15 35 96 47 38 79 23 54 68 09 80 35 45 95 86 17 3 4 5 3 4 5 3 3 3 3 4 4 4 4 4 4 4 5 5 5 5 6 6 6 6 6 6 76 96 9 0 1 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 65 75 7 8 x el e 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 1 5 5 1 5 5 15 pu ma 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- D N D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO02 O 52 2-103121 : .o Ntekco D
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Figure imgf000345_0001
as a g s a s g s s a c sa a s a s a c sg s c a s a s a a a a a a a a s u s sa g s s c s s u s s a s s g s s s u s c s a s a s a s u s u s a u s s a a a a s s a s c s u s s a u s a a s c s s s a s a s a a s u s us u s ' a c 3 u c u c g gu a c ua ag ca u c a g aa u c g u au au u c aa u a g c u u cu c c a g a c t a u g a u c a u a a g u c c a a u a uc ua u c c o u u u u a ' u c c c gc cg a u g ag c cu a a c uu u a gu ca u ca a a u c 5 aa u c u u g u c c a c a ua u ag u a c u u g u c u u u c u a c ag aa u uc u c c u u u u c u g a c c a a u a u u u g g u a a g g u a a u g ec a f f U f u f f f f f f g f c f a f f a U f C f U f U f f u U f f u U f f f a f f C f u C f G C f f a f f u A A G f A f A f G f A f u C f u f u f u f ne U f G U U U C f f U U U f C f C f U f f Cf A G f U f f U f U f C f U f u U q f f f f f f A A C f fC G f C f A f U f U f U f A f f A f U f C f A f C f C f S u G a U g U U u u Uc U u U u u Cc f Gc G U G u U A U C u C U U G C e u f f f f u f f f f f f f f a f a f u f f u f a f u f f a f u f a f u f g f d A ) C )a U )a A )a C )a G )a U )a U )a U )a U )a U )a U )a C )a A )a G ) C )a U )a U )a C )a U )a U )a G )a C )a C )a A ) U ) n a d d d d d d d d d d d a d d d a a ar d d tS A ( U a (u C ( U u ( c U (u A ( G u ( U ( C ( U U ( U ( U ( d G d d d d ( A ( U ( G ( U ( U ( A ( U d d d U U A ( d c a g u ( c u c u u a a a u u u a ( c ( g ( c a C d ( A a ( c es a a a u a u u g u uc a u g u u uc u g g g g g u g g a g c g a g g a a a n c s e S c s s s a s s s u s s u s s s u s g s a s u s u s g s c s s s s s s a s a s s a g s u a s a a s u a s c u s u a s c u s a u s u g s a u s u g s c u s u u s c g s u c g s c s a u s c a s g a s u a s a u s u u s a g s a u s c a s a 87 88 98 09 19 39 49 5 6 7 8 9 0 1 3 9 4 3 7 2 5 6 0 8 3 4 3 3 3 3 3 3 3 93 93 93 93 93 04 54 54 64 74 84 94 3 4 8 9 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 1 1 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 e 5 ma 2 5 2 5 2 5 2 5 2 5 1 2 5 1 2 5 1 2 5 1 2 5 1 2 5 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 - D - D - D - 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 D - D - D - D - D - D - D - D - D - D - D - - - - - - - - - - - - N A A A A A A A A A A A A A A D A D A D A D A D A D A D A D A D A D A D A D A 62 72 82 92 03 13 23 33 43 53 63 73 8 9 0 1 2 3 4 5 6 7 8 9 0 1 : 8 8 8 8 8 8 8 8 8 8 8 8 38 38 48 48 48 48 48 48 48 48 48 48 58 58
Figure imgf000346_0001
as a a s a s a a s a s a s s c s a s a s s a s a s a a a s a a s s a u s a c sa a s a s a s a s a s a g s a a g s s a a su a s a s a g s a s s g s c a sa '3 u c a a a aa g u u a s s a s s s s a s a s a s u s s s a s sa s c s a u sg s a a g u u a u g g au a au ua c a c a a a ua gu gg ca u c u a u cg t u a u a c g u ua u a a aa aa g a u a c a a cu ua a u o ag g aa ga uu u a c u u ' u ug a a u u u u u c a c g u a c a u a u c g ug ua c a a g u a c ug a a u g u a u a u c a u a c uu a u a a a a ua 5 u u g u a a u u c a g a a a u c u u a u u g u a a u u a a a f g f a af a ec c f c ne U f f A f f c f u f u f u f f a f a f f c g u G f a f a f u f g f f u f u f a f f G A f C f C A u f U f A f A f f U A U G f A f U U f U f G f A f u C f U f U f f U f G f A f f f C G A f f G f A f A f U f f f A A f U A f G f f U f f f f C f U f G f A f U f A f A f A f f A f U f G A f U U f G f q A S a C a C a G A a a U U A Ac f A a U A A C U A A U G a G A g c f c f G U e f f f u f f f a f c f a f f a f u f a f a f c f a f a f a f f a f f u f g f d G )a A )a U )a C )a A )a A )a G )a G ) G a U ) )a C )a A )a A ) A )a A )a A )a A )a A )a A ) A )a G )a A )a U )a U )a C )a U ) n d d d d d a d d d a d a d d d d d a ar tS U ( G u (u C ( d a A ( U u (g A ( d d u ( c A d d d d d d U d ( A G u ( a (g G (g C ( dA U A ( A A A d ( A G ( A ( G ( U ( G ( U ( C ( g g ( c (a u (a ( c a ( c a u a a u a g u es c c u a g c u g u g c g c u u u g g g g a a u u a u ac g g a g g uc a g n s c e g s s a s u s s a s s g s s s s s c s a a s s s s g s u s s u s s u s a s S s u u u s c s u a s g g s c u s u g s c a s a g s g a s a u s u a s g s u s u g s g c s a g s c u s a g s u u s a a s a g s g u s g a s u g s c u s u u s c 95 86 17 37 49 59 30 4 5 4 5 6 7 8 9 0 9 0 1 8 0 1 4 7 8 9 6 6 6 6 6 6 7 17 08 28 28 28 28 28 28 38 38 48 48 8 9 9 3 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 8 8 8 9 9 9 9 1 1 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 5 5 e 5 ma 2 5 2 5 2 5 2 5 2 5 1 2 5 1 2 5 1 2 5 1 2 5 1 2 5 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 - D - D - D - 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 D - D - D - D - D - D - D - D - D - D - D - - - - - - - - - - - - N A A A A A A A A A A A A A A D A D A D A D A D A D A D A D A D A D A D A D A 25 35 45 55 65 75 85 95 06 16 26 36 46 56 66 76 8 9 0 1 2 3 : 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 68 68 78 78 78 78
Figure imgf000347_0001
n uP u u u u u u u V P V P V P u u u u u u u u u u u u u u A V P V P V P V P V P V P V P V P V P V P V P V P V P V P V P V P V P V P V 08 18 28 38 48 58 68 78 88 98 09 19 29 39 4 5 6 7 8 9 0 1 : 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 0 0 O1 1 11 11 11 1 8 8 8 8 8 8 8 8 8 8 9 9 N 1 1 1 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 as a a a s a a a a s a s a a a a a u s s u s s u s u s a a s a s a s a s a s s u s a u g s s a s su a s a s s s u s c ' a 3 c u u su su u sa u sg a su u s u s s s su u u s c s s c a s u s u s s su g a u u u a a g au ua uu uu a u ua u c u u c a uu uu g ot u c a g a c u uu aa a a g u au u a a a u u u c aa ca u c u c a u u a g u ua g a u c a a a g g a c c c a a a a g u aa gu u g a a u c u c u u '5 a ec a f u a a u f u f a f g u a u f u f a a c u a a a aa a a a g af a a a c a u f a f a a g a af u a g a a a u c c a f u f a f g f a f uc g f a f n A f A eu A f f A A f U f A f A f f f u f A f C f f A f A G A f A f f U G A U U f f A f U f f f U A f f f U U A f U f A A f A f A f A f f U G A f G f q G A A A f A f U G f U f C f A fC U f A f f A G f A fC f A U f A f A G G eS u f g f a f a f a f u A G U u Ac f U a g f u f U a g c f a u u a f a f d G n a U a a A f a f a f g f f f a ) C f f f f f ) ) G ) ) A a C )a C ) A ) A ) G ) U )a C )a A )a )a U ) A )a G )a C )a A )a A )a U )a A )a a d d d d rt U S ( c G ( U ( G d ( A d ad a ( C ( G d ad a d d d a d d d d d ( C ( A d d ( A ( G ( U ( C ( U ( d d d A( A ( A ( G ( C ( U ( A ( A ( es u u n g c g s u u u c g gu ga a c g c a c a g uu uu cg g ag aa ga a c u ua s u g u a ac s g a g ac a a c u s u a g u s g s a s g s ac a s e a s s s S s u g s a u s c g s u s u s c a s s a g s c a s s a c s s a s a s c g s s a c s g u s s a s g s c a s s u g g s c s u u s g g s u a s s a g s a a s u 05 15 25 35 45 14 26 37 47 57 6 7 6 6 8 5 6 7 8 9 0 9 9 9 9 9 9 0 0 7 7 3 8 1 5 5 5 5 5 6 7 5 5 5 5 5 0 0 0 0 0 1 1 2 2 2 2 2 2 2 2 1 1 1 1 1 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 61 6 e 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 5 5 5 5 5 5 15 ma - D - D - D - D - D - D - - - - - - - - 2- 2- 2- 2- 2- 2- 2- 2- N A A A A A A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A D A Docket No.: 121301-22520 ALN-511-WO Table 6. Single dose screen for dsRNA agents targeting ACVR1C in SK-MEL-28 cells
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Figure imgf000385_0001
Example 3. Design and Synthesis of Additional dsRNA Duplexes Additional siRNAs were designed, synthesized, and prepared using methods known in the art and described above in Example 2. siRNAs targeting the mouse activin A receptor type 1C (ACVR1C) gene (NCBI refseqID NM_001033369.3, NCBI Gene ID: 269275) or the human ACVR1C gene (NCBI refseqID NM_145259.3, NCBI Gene ID: 130399) were designed using custom R and Python scripts. The human NM_145259.3 mRNA has a length of 8853 bases. The mouse NM_001033369.3 mRNA has a length of 8666 bp. A detailed list of the additional unmodified sense and antisense strand sequences of ACVR1C dsRNA agents comprising an unsaturated C22 hydrocarbon chain conjugated to position 6 on the sense strand, counting from the 5’-end of the sense strand are shown in Table 7. A detailed list of the addition modified sense and antisense strand sequences of ACVR1C dsRNA agents comprising an unsaturated C22 hydrocarbon chain conjugated to position 6 on the sense strand, counting from the 5’-end of the sense strand are shown in Table 8.
Docket No.: 121301-22520 ALN-511-WO de tag uj D I 89 99 00 10 20 30
Figure imgf000386_0001
_ _ _ _ 2 e U U M M M M M g r CC G G U CA C N N N N N C a t A U U C U U de A U G G U A U U G A G C U 2 3 4 5 t ar N R U G U U U U G 88 8 8 8 4 8 8 8 u m U U G C U U U U U C 1 41 41 41 t G
Figure imgf000386_0002
U U U C G A A U A A U A n q a e u f g f a f a f u af ac f A A A U G U A N r t S G f U f A f C f C f A d U U G U A f C G G A U G A A G A R s S e na a f u U f u f a a G U f f A u f U A G A A d s r t a a Ag u A U U U U U C n S a a ug a u ag 1 eS e s us gs a as g a 3 R e n f f s G U f f s A f s f 6 2 V h ei s s s G s A s A s 3 4 8 C t t n uP uP u u u u 3- 5 1 6 9 - 5 3 8 A fo A V V P V P V P V P V 6 - - 0 5- f 3 54 93 48 83 o d s 6 7 8 9 e n e D I . 8 8 8 8 09 19 6 7 8 9 c -’ QO8 8 8 8 8 8 7 7 7 7 n 5 E S N41 4 4 4 4 4 8 e e 1 1 1 1 1 4 84 84 8 u 1 1 1 41 q e h t S m a a s a s a s d o a s s r a f c s u c s a s u u s a s c s uc u s c u c A A A A na g ' u u a u c a u A U C A U 3 ua u c a u au u C U U U U r t ni o t a a u a U C U C C S t ' u a c ag uu g C A n a a u u U A U C e 5 c u g A U A U s u a f c f u f f g f A A U U A A C A U G n A U G U e o e C c n A f U f C f G f u f Af U f C U U G U s G i t , e d u G f U f U f U f G f C U G A f U U C G U U n U U U A A n q A a e a f u f a f u f A af u f C U G A G S U A U A U U d r t d U ) U ) G a )a U )a C ) U )a U G A U U A n S n ad d d d ad d C U a e a C A U ( U C U G G U U C U A C e s sn r tS ( c ( a g g ( u ( c a ( c U U C U U n e e u u g u g u u G G U G e S s s s s u s a s g s U U G AC U S U U U G e n a d u u g c u G e ei h t S s u s g s u s u s u s g fi no e 31 95 06 1 2 d 6 m 4 3 9 0 1 2 9 4 4 64 64 o a 9 1 5 6 6 6 5 0 0 0 0 n N 85 95 4 4 4 4 3 1 1 1 1 M o 3 3 01 01 0 0 7 4 4 4 4 . i t x 7 7 4 4 14 1 1 2 2 2 2 8 i s e l 1 1 2 2 4 - - - - - e l p - - - - 2- 2- D D D D D b o P u D D D D D D A A A A A a D A A A A A A T o t Docket No.: 121301-22520 ALN-511-WO Example 4. In vivo Screening of dsRNA Duplexes in Mice The dsRNA agents targeting ACVR1C listed in Tables 7 and 8 were further evaluated for efficacy in vivo. In the initial set of experiments, the efficacy of AD-1735913 to inhibit the expression of ACVR1C was evaluated in vivo. At day 0, groups of three mice were subcutaneously administered a single dose of AD- 1735913 at either 0.5 mg/kg, 2 mg/kg, 5 mg/kg, or PBS control (Study Group 1) (see Table 9). At day 21 post-dose animals were sacrificed, adipose samples were collected and snap-frozen in liquid nitrogen. mRNA was extracted from subcutaneous and gonadal white adipose as well as brown adipose tissues and analyzed by the RT-qPCR method. ACVR1C mRNA levels were compared to housekeeping gene PPIA. The values were then normalized to the average of PBS vehicle control group. The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results, shown in FIG 1, demonstrate that AD-1735913 potently reduced the level of the mouse ACVR1C messenger RNA in vivo in white and brown adipose tissues. Table 9. Treatment Groups
Figure imgf000387_0001
In a separate group of experiments, additional duplexes were evaluated for in vivo efficacy. Mice were subcutaneously administered a single dose of 5 mg/kg of the indicated duplexes or PBS control (see Table 10). At day 21 post-dose animals were sacrificed, adipose samples were collected and snap-frozen in liquid nitrogen. mRNA was extracted from white adipose and brown adipose tissues. Serum samples were also obtained. ACVR1C mRNA levels were determined as described above. The results are shown in FIG.2. These data demonstrate that subcutaneous administration of a single 5 mg/kg dose of the indicated dsRNA agents potently inhibited ACVR1C mRNA in white and brown adipose tissues and that a single dose of AD-2410459.1 and AD-2410460.1 resulted in about 70% knockdown of ACVR1C mRNA levels in both gonadal white adipose tissue and subcutaneous white adipose tissue. Docket No.: 121301-22520 ALN-511-WO Table 10. Treatment Groups
Figure imgf000388_0001
Example 5. Design and Synthesis of Additional dsRNA Duplexes and in Vitro Screening Additional siRNAs were designed, synthesized, and prepared using methods known in the art and described above in Example 2. siRNAs targeting the human activin A receptor type 1C (ACVR1C) gene (NCBI refseqID NM_145259.3, NCBI Gene ID: 130339) were also designed using custom R and Python scripts. The human NM_145259.3 mRNA has a length of 8853 bases. A detailed list of the additional unmodified sense and antisense strand sequences of ACVR1C dsRNA agents comprising an unsaturated C22 hydrocarbon chain conjugated to position 6 or position 16 on the sense strand, counting from the 5’-end of the sense strand are shown in Table 11. A detailed list of the addition modified sense and antisense strand sequences of ACVR1C dsRNA agents comprising an unsaturated C22 hydrocarbon chain conjugated to position 6 or position 16 on the sense strand, counting from the 5’-end of the sense strand are shown in Table 12. Culture of SK-MEL-28 cells (Cat # HTB-72, ATCC, Manassas, VA), transfections, total RNA isolation, cDNA synthesis, RT-PCR, and calculations of relative fold change were performed as described in Example 2. Transfection assays were performed at 10 nM, 1 nM, 0.1 nM, 0.001 nM, and 0.0001 nM. Free uptake assays in SK-MEL-28 cells were performed similarly to the transfection assays but without the use of Liporectamine RNAimax and cells were incubated for 48 hours prior to the RNA purification. Free uptake assays were performed at 2000 nM, 1000 nM, 100 nM, and 10 nM. Table 13 shows the results of the single dose screens in SK-MEL-28 cells transfected with the indicated agents from Tables 11 and 12 and the results of the single dose screens in SK-MEL-28 cells by free uptake with the indicated agents from Tables 11 and 12. Docket No.: 121301-22520 ALN-511-WO 3. 95 i 2 77 51 51 4 7 9 3 4 n 5 8 5 1 7 5 n 4 1 6 3 7 7 6 3 3 7 3 3 i e 41 3 2 6 1- 1- 1- 1- 1- 2 1- 1- 1-
Figure imgf000389_0001
mS e s G C A G A A G C C C C U o C e i t A A U G A A U U U A U U n A C G A U U U U A A st h t C A f A U U U U U U U U U U U U ne o g d An e A-’ N5 3 . R e 9 s h t 52 77 51 51 4 7 9 3 4 d n i 5 e 4 43 12 66 3 1 7 7 86 53 3 1 7 5 2 7 3 3 C mo g 1_ 3- 3- 4 - 1- 1- 1- 1- 3 1- 1- 1- 1 r f na M 4 1 - 6 75 59 59 46 73 - 3 99 35 43 R Vg R N 13 03 44 31 61 61 61 31 03 61 31 31 C ni Atn 40 50 60 70 80 90 01 11 2 3 4 5 f u Q 9 9 9 9 9 9 1 1 1 1 o o E O4 4 4 4 9 9 9 9 9 9 se C, S D I N 1 1 1 1 41 41 41 41 41 41 41 41 c n d e n u a qe r t S A A A A S A A A A A G A A A U U d e s U U A A A G U na n e U U C A C U U A A U A A G A C A G S C U U U C G G G A rt A U U U A A U U U U U C S e es h A G t G G A A G A G A U A ’ A U U A U U U A U U U U n n 3 C U G A A G C G U C A U U A U e G G A U A U G s o it 6 o t 1 ’ U U A U U U U A A U 5 C C C U U U U A U A U U A A A C U U U n n e c G G G U A A G U A C Ao d i G t n C U U U A U C G A U A U C A A A A A U U G U n i a s e G A U U o u A A C G G A U C q U U U A U U G G es P e U U U G S G G G A C C C U A U U U n r U U G C C A e o e s A U C U A A G G U G G A G G A A S 6 n U A e U U A G A A U U U U G A d n S U G C U A A A A G C U G ei o fi i t d i s o o m P n o t e m 05 67 91 19 49 25 35 75 85 06 1 2 Ud a 1 1 3 1 4 0 0 0 0 6 6 N 8 8 8 9 9 0 0 0 0 0 0 0 . e t 0 0 0 0 0 4 4 4 0 0 0 1 a x 5 5 5 5 5 6 6 4 4 4 4 1 g e l 2 2 2 2 2 6 6 6 6 6 el u b j p - - - - - 2- 2- 2- 2- 2- 2- 2- u D D D a n D D D D D D D D D o D A A A A A A A A A A A A T C Docket No.: 121301-22520 ALN-511-WO 2 Q 5 35 45 55 65 7 8 9 0 1 2 3 : 5 5 5 6 6 6 6 E S D I O9 N4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 4 9 1 41
Figure imgf000390_0001
V n C nu s s e f f gs s s s s s s s s s A f C G f A f U f U f U f U f A f s f f Ao i t su su su su su su su su su A su C s A s f C n P P P P P P P P P u u o , A V V V V V V V V P P P s d V V V V ec n n a 8 e r t : 2 92 03 13 23 33 43 5 6 7 8 9 u 9 9 9 9 3 3 3 3 3 S QE e s D I O N4 4 4 4 94 94 94 94 94 94 94 94 q e S 1 1 1 1 1 1 1 1 1 1 1 1 S n d e n S a e a s a s a r t h t u s a s a s s g s a s a a s a s a s a s a c u s s a s a s a s a s u s u s g s s u S n u c ug sa c c u u a e c a a g g ug a s g aa s o aa ug uu u u g a u a aa ug u u a g u ) u ) c ) n 6 es 1 a c uu gu a u u u a g a a g a c ug ad ad ad it n u u u ’ u n oi t 3 f u C f a f u g g u a u u C f u f A ( U ( A ( f c f f u f u f u f u Af U u u c ua Ai s o t d o U f U A f U f C A f A f U f G f U a af u a g u n P ’5 U f G A f f C U G f G f C f f f f f A U C a r U A e a f u f c f a f U af U af a f a f u f f U A f f e o c n G ) A ) U ) U ) A ) A C ) G ) G ) f A G f A G f s 6 e a a e a a n a )a a a a U n u d d d d d d d u f g f u f S o q U ( U ( U ( A ( d d U U U U ( U ( Ag Ug Cg d i t e i S gu gu g a ( ( ( g c c c c cu u c a u ua u ei s fi e d P n g g a u a g u g a a o s s u s u sa s g g g s s s s s a s a a u s s o o t e S s u u s g s c s u a s a a s a u s a u s a u s g g s c s u a s g Md . e t 05 67 9 1 4 2 3 7 8 0 1 2 2 a x 1 1 1 9 9 5 5 5 5 6 6 6 1 g e e l e 3 1 4 0 0 0 0 0 0 0 l u j p m- 80 - 80 - 80 - 90 - 90 - 04 - 04 - 04 - 04 - 04 - 04 - 04 b n u a D D5 D5 D5 D5 D5 D6 D6 D6 D6 D6 D6 D6 a o N A 2 A 2 A 2 A 2 A 2 A 2 A 2 A 2 A 2 A 2 A 2 A 2 T C Docket No.: 121301-22520 ALN-511-WO 39 20 1 1 0 6 1 9 70 9 1 0 5 1 0 1 1 0 9 1 0 5 1 1 0 1 0 6 1 0 9 1 01 mn 01
Figure imgf000391_0001
- L 23 73 43 25 54 66 64 34 54 33 52 04 73 E m M- n 1 K 0 G S 0 . V ni 0 A C 03 73 13 54 34 46 24 14 94 53 13 63 5 1 3 R V mn C 1 G A 0 g . V 0 A ni te 72 23 72 63 83 15 04 63 93 82 0 1 1 gr 3 3 3 a T mn s G t 1 . V ne 0 A g no A i t 22 92 02 13 62 93 72 5 8 0 3 5 4 c 2 3 2 2 2 2 A e N f s Rs n G d a r mn V f T 1 A o 8 n 2 e - 6 5 4 9 7 2 6 6 4 4 3 6 7 e L 1 1 1 1 1 2 1 1 2 1 1 1 1 rc E S M- mn G es K0 V o S 1 A D elg e ni m 2 3 0 1 a 50 50 6 6 9 1 7 8 2 4 0 3 0 9 7 1 6 5 5 6 9 5 1 S N 0 0 1 1 3 0 0 0 0 4 1 9 . 4 4 04 9 8 8 0 0 0 0 9 8 5 3 x 6 6 0 0 0 4 4 4 4 0 0 3 1 e l 2 6 5 5 5 6 6 6 6 5 5 7 el p - 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 2- 1- b u D D D D D D D D D D D D D a D A A A A A A A A A A A A A T Docket No.: 121301-22520 ALN-511-WO Example 6. In vivo Screening of dsRNA Duplexes in Non-Human Primates Duplexes of interest targeting ACVR1C were further evaluated for efficacy in vivo in non- human primates (NHP). At day 1, groups of 3 cynomolgus monkeys (Macaca fascicularis) were subcutaneously administered a single 3 mg/kg dose of AD-2640052, AD-2640053, AD-2640060, AD-2509191, or AD-2508176, or PBS control. At days -15 and -2 (pre-dose), and days 15, 28, 43, 57, 71, and 85 post- dose subutanous adipose biopsy was performed. Groups dosed with AD-2640052, AD-2640053, AD- 2640060 and PBS were sacrificed on day 85 and various types of adipose tissue samples were collected and snap-frozen in liquid nitrogen. The types of terminal adipose tissue collected included abdominal adipose tissue, white mesenteric adipose tissue, brown adipose tissue, inguinal adipose tissue, perirenal adipose tissue, gonadal adipose tissue, and periaortic adipose tissue. mRNA was extracted from the tissue and analyzed by the RT-qPCR method. ALK7 protein in monkey adipose was quantified via liquid chromatography-mass spectrometry (LC-MS). The signature peptide of ALK7 protein selected for quantification was TIVLQEIVGK (SEQ ID NO: 14966). The peak area ratio of the signature peptide versus that of the internal standard was used as a proxy for the abundance of ALK7 protein in each sample. The approximate ALK7 percent protein remaining was calculated based on the peak area ratio of each sample relative to the average peak area ratio of Day -15 and Day -2 of the same animal. The mRNA results are presented in Table 14 and FIG.3 for biopsy, in Table 15 and FIG. 4 for Day 85 terminal adipose samples. ACVR1C mRNA levels were normalized to the average of PBS vehicle control group. Two housekeeping genes were also used as controls (ELF1 and RPL30). The data were expressed as percent of baseline value, and presented as mean plus standard deviation. The results in Table 14 and Table 15 demonstrate that AD-2640052, AD-2640053, and AD-2640060 potently and durably inhibit the expression of ACVR1C messenger RNA in vivo in all of adipose tissues examined. AD-2640052, AD-2640053, AD-2640060, AD-2509191, and AD-2508176 also result in the reduction of ALK7 protein, as shown in FIG.5, FIG.6, and FIG. 7. Table 14.
Figure imgf000392_0001
Docket No.: 121301-22520 ALN-511-WO
Figure imgf000393_0001
Docket No.: 121301-22520 ALN-511-WO
Figure imgf000394_0001
Docket No.: 121301-22520 ALN-511-WO
Figure imgf000395_0001
Table 15.
Figure imgf000395_0002
Docket No.: 121301-22520 ALN-511-WO
Figure imgf000396_0001
Docket No.: 121301-22520 ALN-511-WO EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

Docket No.: 121301-22520 ALN-511-WO We claim: 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2 and wherein one or more C22 hydrocarbon chains are conjugated to the sense strand. 2. The dsRNA agent of claim 1, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 3. The dsRNA agent of claim 1, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than two nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 4. The dsRNA agent of claim 1, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than one nucleotide from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 5. The dsRNA agent of claim 1, wherein the antisense strand comprises at least 15 contiguous nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 6. The dsRNA agent of claim 1, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 7. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding ACVR1C, and wherein the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 8. The dsRNA agent of claim 7, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than two nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. Docket No.: 121301-22520 ALN-511-WO 9. The dsRNA agent of claim 7, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than one nucleotide from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 10. The dsRNA agent of claim 7, wherein the 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 2-5, 7, 8, 11, and 12. 11. The dsRNA agent of claim 7, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the sense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the antisense strand nucleotide sequences in any one of Tables 2-5, 7, 8, 11, and 12. 12. The dsRNA agent of claim 7, wherein, the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, and AD-2508176. 13. The dsRNA agent of claim 7, wherein, the sense strand and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-2640052, AD-2640053, AD-2640060, AD-2509191, or AD-2508176. 14. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense strand nucleotide sequences selected from the group consisting of (a) 5’-UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’-UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’-UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’-UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. Docket No.: 121301-22520 ALN-511-WO 15. The dsRNA agent of claim 14, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 2 nucleotides from any one of the antisense strand nucleotide sequences selected from the group consisting of (a) 5’-UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’-UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’-UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’-UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. 16. The dsRNA agent of claim 14, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1 nucleotide from any one of the antisense strand nucleotide sequences selected from the group consisting of (a) 5’-UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’-UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’-UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’-UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. 17. The dsRNA agent of claim 14, wherein the antisense strand comprises at least 15 contiguous nucleotides from any one of the antisense strand nucleotide sequences selected from the group consisting of (a) 5’-UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’-UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’-UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’-UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. 18. The dsRNA agent of claim 14, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) 5’- AAGCCUAAUGAUGAUAAUUAA-3’ of SEQ ID NO: 14909 and 5’- UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-AUAUCUCAACUUUGUGUCAAA-3’ of SEQ ID NO: 14910 and 5’- UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’- CUAAUGAUGAUAAUUAUGUUA-3’ of SEQ ID NO: 14913 and 5’- UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’- UGUGAAUAUCUUUGAGUCCUA-3’ of SEQ ID NO: 7805 and 5’- UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and Docket No.: 121301-22520 ALN-511-WO (e) 5’- GUGUGUAUGUCUUUUGUGUGA-3’ of SEQ ID NO: 7470 and 5’- UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. 19. The dsRNA agent of claim 14, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 2 nucleotides and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 2 nucleotides from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) 5’- AAGCCUAAUGAUGAUAAUUAA-3’ of SEQ ID NO: 14909 and 5’- UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-AUAUCUCAACUUUGUGUCAAA-3’ of SEQ ID NO: 14910 and 5’- UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’- CUAAUGAUGAUAAUUAUGUUA-3’ of SEQ ID NO: 14913 and 5’- UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’- UGUGAAUAUCUUUGAGUCCUA-3’ of SEQ ID NO: 7805 and 5’- UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’- GUGUGUAUGUCUUUUGUGUGA-3’ of SEQ ID NO: 7470 and 5’- UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. 20. The dsRNA agent of claim 14, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 1 nucleotide and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1 nucleotide from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) 5’- AAGCCUAAUGAUGAUAAUUAA-3’ of SEQ ID NO: 14909 and 5’- UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; (b) 5’-AUAUCUCAACUUUGUGUCAAA-3’ of SEQ ID NO: 14910 and 5’- UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’- CUAAUGAUGAUAAUUAUGUUA-3’ of SEQ ID NO: 14913 and 5’- UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’- UGUGAAUAUCUUUGAGUCCUA-3’ of SEQ ID NO: 7805 and 5’- UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’- GUGUGUAUGUCUUUUGUGUGA-3’ of SEQ ID NO: 7470 and 5’- UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. 21. The dsRNA agent of claim 14, wherein the sense strand comprises at least 15 contiguous nucleotides and the antisense strand comprises at least 15 contiguous nucleotides from any one of the sense and antisense strand nucleotide sequences selected from the group consisting of (a) 5’- AAGCCUAAUGAUGAUAAUUAA-3’ of SEQ ID NO: 14909 and 5’- UUAATUAUCAUCAUUAGGCUUUG-3’ of SEQ ID NO: 14921; Docket No.: 121301-22520 ALN-511-WO (b) 5’-AUAUCUCAACUUUGUGUCAAA-3’ of SEQ ID NO: 14910 and 5’- UUUGACACAAAGUUGAGAUAUAG-3’ of SEQ ID NO: 14922; (c) 5’- CUAAUGAUGAUAAUUAUGUUA-3’ of SEQ ID NO: 14913 and 5’- UAACAUAAUUAUCAUCAUUAGGC-3’ of SEQ ID NO: 14925; (d) 5’- UGUGAAUAUCUUUGAGUCCUA-3’ of SEQ ID NO: 7805 and 5’- UAGGACUCAAAGAUAUUCACAUU-3’ of SEQ ID NO: 9291; and (e) 5’- GUGUGUAUGUCUUUUGUGUGA-3’ of SEQ ID NO: 7470 and 5’- UCACACAAAAGACAUACACACUU-3’ of SEQ ID NO: 8956. 22. The dsRNA agent of any one of claims 1-21, wherein at least one nucleotide of the dsRNA agent comprises a nucleotide modification. 23. The dsRNA agent of any one of claims 1-22, wherein substantially all of the nucleotides of the sense strand comprise a nucleotide modification; substantially all of the nucleotides of the antisense strand comprise a nucleotide modification; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a nucleotide modification. 24. The dsRNA agent of any one of claims 1-23, wherein all of the nucleotides of the sense strand comprise a nucleotide 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 s. 25. The dsRNA agent of any one of claims 22-24, wherein at least one of the nucleotide modifications is selected from the group consisting of a deoxy-nucleotide modification, a 3’-terminal deoxythimidine (dT) nucleotide 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’-methoxyethyl nucleotide modification, a 2’-O-alkyl- nucleotid modification e, a morpholino nucleotide modification, a phosphoramidate modification, a non-natural base comprising nucleotide modification, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexenyl nucleotide modification, a nucleotide comprising a phosphorothioate group modification, a nucleotide comprising a methylphosphonate group modification, a nucleotide comprising a 5’-phosphate modification, a nucleotide comprising a 5’-phosphate mimic modification, a thermally destabilizing nucleotide modification, a glycol modified nucleotide (GNA) modification, a nucleotide comprising a 2’ phosphate modification, and a 2-O-(N-methylacetamide) nucleotide modification; and combinations thereof. Docket No.: 121301-22520 ALN-511-WO 26. The dsRNA agent of any one of claims 22-24, wherein at least one of the nucleotide modifications is selected from the group 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. 27. The dsRNA agent of any one of claims 22-24, wherein at least one of the nucleotide modifications is selected from the group consisting of a deoxy-nucleotide modification, a 2'-O- methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy- nucleotide modification, a glycol nucleotide (GNA) modification, a nucleotide comprising a 2’ phosphate modification, a nucleotide comprising a phosphorothioate group modification, and a vinyl- phosphonate nucleotide modification; and combinations thereof. 28. The dsRNA agent of any one of claims 22-24, wherein at least one of the nucleotide modifications is a nucleotide modified with a thermally destabilizing nucleotide modification. 29. The dsRNA agent of claim 28, wherein the thermally destabilizing nucleotide modification is 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). 30. The dsRNA agent of any one of claims 22-24, wherein the antisense strand comprises 2'- fluoro nucleotide modifications at positions 2, 14, and 16 counting from the 5’ end. 31. The dsRNA agent of any one of claims 22-24, wherein the antisense strand comprises 2'- fluoro nucleotide modifications at positions 2, 6, 14, and 16 counting from the 5’-end. 32. The dsRNA agent of any one of claims 22-24, wherein the antisense strand comprises 2'- fluoro nucleotide modifications at positions 9-11 counting from the 5’-end. 33. The dsRNA agent of any one of claims 1-32, further comprising a phosphate or phosphate mimic at the 5’-end of the antisense strand. 34. The dsRNA agent of claim 33, wherein the phosphate mimic is a 5’-vinyl phosphonate (VP). 35. The dsRNA agent of any one of claims 1-34, wherein 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, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl. Docket No.: 121301-22520 ALN-511-WO 36. The dsRNA agent of any one of claims 1-35, wherein the double stranded region is 19-30 nucleotide pairs in length. 37. The dsRNA agent of claim 36, wherein the double stranded region is 19-25 nucleotide pairs in length. 38. The dsRNA agent of claim 36, wherein the double stranded region is 19-23 nucleotide pairs in length. 39. The dsRNA agent of claim 36, wherein the double stranded region is 23-27 nucleotide pairs in length. 40. The dsRNA agent of claim 36, wherein the double stranded region is 21-23 nucleotide pairs in length. 41. The dsRNA agent of any one of claims 1-40, wherein each strand is independently no more than 30 nucleotides in length. 42. The dsRNA agent of any one of claims 1-41, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. 43. The dsRNA agent of any one of claims 1-42, wherein the region of complementarity is at least 17 nucleotides in length. 44. The dsRNA agent of any one of claims 1-43, wherein the region of complementarity is between 19 and 23 nucleotides in length. 45. The dsRNA agent of any one of claims 1-44, wherein the region of complementarity is 19 nucleotides in length. 46. The dsRNA agent of any one of claims 1-45, wherein at least one strand comprises a 3’ overhang of at least 1 nucleotide. 47. The dsRNA agent of any one of claims 1-46, wherein at least one strand comprises a 3’ overhang of at least 2 nucleotides. 48. The dsRNA agent of any one of claims 7-47, wherein one or more C22 hydrocarbon chains is conjugated to one or more internal positions on at least one strand. Docket No.: 121301-22520 ALN-511-WO 49. The dsRNA agent of any one of claims 1-6 and 48, wherein the C22 hydrocarbon chain is saturated or unsaturated. 50. The dsRNA agent of any one of claims 1-6, 48, and 49, wherein the C22 hydrocarbon chain is linear or branched 51. The dsRNA agent of any one of claims 48-50, wherein the internal positions include all positions except two or three terminal positions from each end of the at least one strand. 52. The dsRNA agent of claim 51, wherein the internal positions exclude a cleavage site region of the sense strand. 53. The dsRNA agent of claim 51, wherein the internal positions exclude positions 9-12 or positions 11-13, counting from the 5’-end of the sense strand. 54. The dsRNA agent of claim 51, wherein the internal positions exclude a cleavage site region of the antisense strand. 55. The dsRNA agent of claim 54, wherein the internal positions exclude positions 12-14, counting from the 5’-end of the antisense strand. 56. The dsRNA agent of any one of claims 48-55, wherein the one or more C22 hydrocarbon chains are conjugated to 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. 57. The dsRNA agent of claim 56, wherein the one or more C22 hydrocarbon chains are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, 16, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’-end of each strand. 58. The dsRNA agent of claim 57, wherein the one or more C22 hydrocarbon chains are conjugated to position 6 on the sense strand, counting from the 5’-end of the sense strand. 59. The dsRNA agent of claim 57, wherein the one or more C22 hydrocarbon chains are conjugated to position 16 on the sense strand, counting from the 5’-end of the sense strand. Docket No.: 121301-22520 ALN-511-WO 60. The dsRNA agent of any one of claims 1-6 and 48-59, wherein the one or more C22 hydrocarbon chains is an aliphatic, alicyclic, or polyalicyclic compound. 61. The dsRNA agent of claim 60, wherein the one or more C22 hydrocarbon chains contains a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. 62. The dsRNA agent of any one of claims 1-6 and 48-61, wherein the one or more C22 hydrocarbon chains is a C22 acid. 63. The dsRNA agent of claim 62, wherein the C22 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. 64. The dsRNA agent of any one of claims 1-6 and 48-61, wherein the one or more C22 hydrocarbon chains is a C22 alcohol. 65. The dsRNA agent of claim 64, wherein the C22 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. 66. The dsRNA agent of any one of claims 1-6 and 48-61, wherein the one or more C22 hydrocarbon chains is a C22 amide. 67. The dsRNA agent of claim 66, wherein the C22 amide is selected 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. 68. The dsRNA agent of any one of claims 1-6 and 48-67, wherein the one or more C22 hydrocarbon chains is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s). 69. The dsRNA agent of claim 68, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, Docket No.: 121301-22520 ALN-511-WO isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone. 70. The dsRNA agent of any one of claims 1-6 and 48-69, wherein the one or more C22 hydrocarbon chains is conjugated to the 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, or carbamate. 71. The dsRNA agent of any one of claims 1-6 and 48-70, wherein the one or more C22 hydrocarbon chains is conjugated to the dsRNA agent via a linker or a carrier or via internucleotide phosphate linkage. 72. The dsRNA agent of any one of claims 1-6 and 48-71, wherein the one or more C22 hydrocarbon chains is conjugated to a nucleobase, sugar moiety, or internucleosidic phosphate linkage. 73. The dsRNA agent of any one of claims 1-72, wherein the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. 74. The dsRNA agent of claim 73, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3’-terminus of one strand. 75. The dsRNA agent of claim 74, wherein the strand is the antisense strand. 76. The dsRNA agent of claim 74, wherein the strand is the sense strand. 77. The dsRNA agent of claim 73, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5’-terminus of one strand. 78. The dsRNA agent of claim 77, wherein the strand is the antisense strand. 79. The dsRNA agent of claim 77, wherein the strand is the sense strand. 80. The dsRNA agent of claim 73, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5’- and 3’-terminus of one strand. 81. The dsRNA agent of claim 80, wherein the strand is the antisense strand. Docket No.: 121301-22520 ALN-511-WO 82. The dsRNA agent of any one of claims 1-81, wherein the base pair at the 1 position of the 5^- end of the antisense strand of the duplex is an AU base pair. 83. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of activin A receptor type 1C (ACVR1C) in a cell, wherein the dsRNA agent comprises a sense strand having 18- 23 nucleotides and at least three 2’-F modifications and an antisense strand having 18-23 nucleotides and four 2’-F modifications, wherein the dsRNA agent has a lipophilic moiety conjugated to the sense strand. 84. The dsRNA agent of claim 83, wherein the antisense strand comprises 2’-F modifications at positions 2, 14, and 16 counting from the 5’-end. 85. The dsRNA agent of claim 83 or 84, wherein the sense strand comprises 2’-F modifications at positions 9-11 counting from the 5’-end. 86. The dsRNA agent of any one of claism 83-85, wherein the lipophilic moiety is a C22 hydrocaron chain attached at position 6 or position 16 on the sense strand counting from the 5’-end. 87. The dsRNA agent of any one of claims 83-86, wherein the dsRNA agent is selected from the group consisting of AD-2509512, AD-2640052, AD-2640053, AD-2640060, AD-2509191, AD- 2509494, AD-2509477, and AD-2508176. 88. A cell containing the dsRNA agent of any one of claims 1-87. 89. A pharmaceutical composition for inhibiting expression of an activin A receptor type 1C (ACVR1C) gene, comprising the dsRNA agent of any one of claims 1-87 and a pharmaceutically acceptable carrier. 90. The pharmaceutical composition of claim 89, wherein dsRNA agent is in an unbuffered solution. 91. The pharmaceutical composition of claim 90, wherein the unbuffered solution is saline or water. 92. The pharmaceutical composition of claim 89, wherein said dsRNA agent is in a buffer solution. 93. The pharmaceutical composition of claim 92, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. Docket No.: 121301-22520 ALN-511-WO 94. The pharmaceutical composition of claim 92, wherein the buffer solution is phosphate buffered saline (PBS). 95. A method of inhibiting expression of an activin A receptor type 1C (ACVR1C) gene in a cell, the method comprising contacting the cell with the dsRNA agent of any one of claims 1-87, or the pharmaceutical composition of any one of claims 89-94, thereby inhibiting expression of the ACVR1C gene in the cell. 96. The method of claim 95, wherein the cell is an adipocyte. 97. The method of claim 95 or 96, wherein the cell is within a subject. 98. The method of claim 97, wherein the subject is a human. 99. The method of claim 97 or 98, wherein the subject has an ACVR1C-associated disorder. 100. The method of claim 99, wherein the ACVR1C-associated disorder is a metabolic disorder. 101. The method of claim 100, wherein the metabolic disorder is metabolic syndrome. 102. The method of claim 100, wherein the metabolic disorder is cardiovascular disease. 103. The method of claim 100, wherein the metabolic disorder is hypertension. 104. The method of any one of claims 95-103, wherein contacting the cell with the dsRNA agent inhibits the expression of the ACVR1C gene by at least 50%, 60%, 70%, 80%, 90%, or 95%. 105. The method of any one of claims 95-104, wherein inhibiting expression of the ACVR1C gene decreases ALK7 protein level in serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%. 106. A method of treating a subject having a disorder that would benefit from reduction in activin A receptor type 1C (ACVR1C) expression, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1-87, or the pharmaceutical composition of any one of claims 89-94, thereby treating the subject having the disorder that would benefit from reduction in ACVR1C expression. Docket No.: 121301-22520 ALN-511-WO 107. A method of preventing at least one symptom in a subject having a disorder that would benefit from reduction in activin A receptor type 1C (ACVR1C) expression, comprising administering to the subject a prophylactically effective amount of the dsRNA agent of any one of claims 1-87, or the pharmaceutical composition of any one of claims 89-94, thereby preventing at least one symptom in the subject having the disorder that would benefit from reduction in ACVR1C expression. 108. The method of claim 106 or 107, wherein the disorder is an ACVR1C-associated disorder. 109. The method of claim 108, wherein the ACVR1C-associated disorder is a metabolic disorder. 110. The method of claim 109, wherein the metabolic disorder is metabolic syndrome. 111. The method of claim 109, wherein the metabolic disorder is cardiovascular disease. 112. The method of claim 109, wherein the metabolic disorder is hypertension. 113. The method of any one of claims 106-112, wherein the subject is a human. 114. The method of any one of claims 106-113, wherein administration of the dsRNA agent to the subject causes a decrease in ALK7 protein accumulation in the subject. 115. The method of any one of claims 106-114, wherein the dsRNA agent is administered to the subject subcutaneously. 116. The method of any one of claims 106-115, further comprising determining the level of ACVR1C in a sample(s) from the subject. 117. The method of any one of claims 106-116, further comprising administering to the subject an additional therapeutic agent for treatment of an ACVR1C-associated disorder. 118. The method of claim 117, wherein the additional therapeutic agent is selected from the group consisting of insulin, a glucagon-like peptide 1 agonist, a sulfonylurea, a seglitinide, a biguanide, a thiazolidinedione, an alpha-glucosidase inhibitor, an SGLT2 inhibitor, a DPP-4 inhibitor, an HMG- CoA reductase inhibitor, a statin, and a combination of any of the foregoing. 119. A kit comprising the dsRNA agent of any one of claims 1-87, or the pharmaceutical composition of any one of claims 89-94. Docket No.: 121301-22520 ALN-511-WO 120. A vial comprising the dsRNA agent of any one of claims 1-87, or the pharmaceutical composition of any one of claims 89-94. 121. A syringe comprising the dsRNA agent of any one of claims 1-87, or the pharmaceutical composition of any one of claims 89-94. 122. An RNA-induced silencing complex (RISC) comprising an antisense strand of any of the dsRNA agents of claims 1-87.
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