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WO2025193754A2 - Compositions et méthodes d'inhibition de l'expression de gènes de la sous-unité bêta de l'inhibine (inhbe) - Google Patents

Compositions et méthodes d'inhibition de l'expression de gènes de la sous-unité bêta de l'inhibine (inhbe)

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Publication number
WO2025193754A2
WO2025193754A2 PCT/US2025/019447 US2025019447W WO2025193754A2 WO 2025193754 A2 WO2025193754 A2 WO 2025193754A2 US 2025019447 W US2025019447 W US 2025019447W WO 2025193754 A2 WO2025193754 A2 WO 2025193754A2
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Prior art keywords
seq
sequence
strand comprises
dsrna
sense strand
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PCT/US2025/019447
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English (en)
Other versions
WO2025193754A3 (fr
Inventor
Kevin J. Kim
Luping LIN
Tianbu ZHANG
Sajesh PARATHATH
Zhou FANG
Pathi PANDARINATHAN
Saul MARTINEZ MONTERO
Jing Yang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Basecure Therapeutics LLC
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Basecure Therapeutics LLC
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Priority claimed from PCT/US2024/019416 external-priority patent/WO2024187190A2/fr
Application filed by Basecure Therapeutics LLC filed Critical Basecure Therapeutics LLC
Publication of WO2025193754A2 publication Critical patent/WO2025193754A2/fr
Publication of WO2025193754A3 publication Critical patent/WO2025193754A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
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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]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—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
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
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    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate

Definitions

  • the disclosure relates to double-stranded ribonucleic acid (dsRNA) targeting INHBE genes, and methods of using the dsRNA to inhibit expression of INHBE in a cell.
  • dsRNA double-stranded ribonucleic acid
  • the present disclosure is based, in part, upon the development of double- stranded ribonucleic acid (dsRNA) targeting Inhibin Subunit Beta E ⁇ INHBE) genes, pharmaceutical compositions comprising the dsRNAs targeting INHBE genes and methods of using the dsRNA to inhibit expression of INHBE in a cell.
  • dsRNA double- stranded ribonucleic acid
  • INHBE Inhibin Subunit Beta E ⁇ INHBE
  • the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 598, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 589; (b) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 599, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 590; (c) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 600, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO
  • the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE, wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 607; (b) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 617, and sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 608; (c) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 618, and the sense strand comprises a sequence that is at least 70% or 80% identical to
  • dsRNA double- stranded ribonucleic acid
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length
  • the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 607
  • the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 617, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 608
  • the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 618, and the sense strand a sequence that is at least 70% or 80% identical
  • the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 598, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589; (b) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 599, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 590; (c) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence
  • the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE, wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 616, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 607; (b) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 617, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 608; (c) the antisense strand comprises at least 15 contiguous nucleotides of an antis
  • the sense strand is 70%, 80%, 90%, 95% or more identical to the sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594,
  • SEQ ID NO: 614 SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682,
  • SEQ ID NO: 683 SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687,
  • the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611 , SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 611 , SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO:
  • the sense strand comprises: (a) 20 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596,
  • SEQ ID NO: 658 SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684,
  • the antisense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ
  • the antisense strand comprises: (a) 21 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO:
  • SEQ ID NO: 697 SEQ ID NO: 698, or SEQ ID NO: 699;
  • 22 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ
  • SEQ ID NO: 698 or SEQ ID NO: 699; and/or (c) 23 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID
  • the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO:
  • the antisense strand is selected from an antisense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO:
  • the antisense strand comprises the sequence of SEQ ID NO: 598 and the sense strand comprises the sequence of SEQ ID NO: 589. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 599 and the sense strand comprises the sequence of SEQ ID NO: 590. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 600 and the sense strand comprises the sequence of SEQ ID NO: 591. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 601 and the sense strand comprises the sequence of SEQ ID NO: 592.
  • the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 593. Tn some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 700. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 603 and the sense strand comprises the sequence of SEQ ID NO: 594. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 604 and the sense strand comprises the sequence of SEQ ID NO: 595.
  • the antisense strand comprises the sequence of SEQ ID NO: 605 and the sense strand comprises the sequence of SEQ ID NO: 596. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 606 and the sense strand comprises the sequence of SEQ ID NO: 597. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 616 and the sense strand comprises the sequence of SEQ ID NO: 607. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 617 and the sense strand comprises the sequence of SEQ ID NO: 608. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 618 and the sense strand comprises the sequence of SEQ ID NO: 609.
  • the antisense strand comprises the sequence of SEQ ID NO: 619 and the sense strand comprises the sequence of SEQ ID NO: 610. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 620 and the sense strand comprises the sequence of SEQ ID NO: 611. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 621 and the sense strand comprises the sequence of SEQ ID NO: 612. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 622 and the sense strand comprises the sequence of SEQ ID NO: 613.
  • the antisense strand comprises the sequence of SEQ ID NO: 623 and the sense strand comprises the sequence of SEQ ID NO: 614. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 624 and the sense strand comprises the sequence of SEQ ID NO: 615. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 665 and the sense strand comprises the sequence of SEQ ID NO: 658. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 666 and the sense strand comprises the sequence of SEQ ID NO: 659.
  • the antisense strand comprises the sequence of SEQ ID NO: 691 and the sense strand comprises the sequence of SEQ ID NO: 682. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 692 and the sense strand comprises the sequence of SEQ ID NO: 683. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 693 and the sense strand comprises the sequence of SEQ ID NO: 684. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 694 and the sense strand comprises the sequence of SEQ ID NO: 685.
  • the antisense strand comprises the sequence of SEQ ID NO: 695 and the sense strand comprises the sequence of SEQ ID NO: 686. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 696 and the sense strand comprises the sequence of SEQ ID NO: 687. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 697 and the sense strand comprises the sequence of SEQ ID NO: 688. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 698 and the sense strand comprises the sequence of SEQ ID NO: 689. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 699 and the sense strand comprises the sequence of SEQ ID NO: 690.
  • the sense strand is 70%, 80%, 90%, 95% or more identical to the sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594,
  • SEQ ID NO: 683 SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687,
  • the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 6
  • the antisense strand comprises: (a) 21 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO:
  • SEQ ID NO: 697 SEQ ID NO: 698, or SEQ ID NO: 699;
  • 22 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 624, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO
  • the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 61 1, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, or SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688
  • SEQ ID NO: 691 SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, or SEQ ID NO: 699.
  • the antisense strand comprises the sequence of SEQ ID NO: 598 and the sense strand comprises the sequence of SEQ ID NO: 589. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 599 and the sense strand comprises the sequence of SEQ ID NO: 590. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 600 and the sense strand comprises the sequence of SEQ ID NO: 591. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 601 and the sense strand comprises the sequence of SEQ ID NO: 592.
  • the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 593. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 700. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 603 and the sense strand comprises the sequence of SEQ ID NO: 594. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 604 and the sense strand comprises the sequence of SEQ ID NO: 595.
  • the antisense strand comprises the sequence of SEQ ID NO: 605 and the sense strand comprises the sequence of SEQ ID NO: 596. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 606 and the sense strand comprises the sequence of SEQ ID NO: 597. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 616 and the sense strand comprises the sequence of SEQ ID NO: 607. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 617 and the sense strand comprises the sequence of SEQ ID NO: 608. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 618 and the sense strand comprises the sequence of SEQ ID NO: 609.
  • the antisense strand comprises the sequence of SEQ ID NO: 619 and the sense strand comprises the sequence of SEQ ID NO: 610. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 620 and the sense strand comprises the sequence of SEQ ID NO: 611. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 621 and the sense strand comprises the sequence of SEQ ID NO: 612. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 622 and the sense strand comprises the sequence of SEQ ID NO: 613.
  • the antisense strand comprises the sequence of SEQ ID NO: 623 and the sense strand comprises the sequence of SEQ ID NO: 614. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 624 and the sense strand comprises the sequence of SEQ ID NO: 615. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 665 and the sense strand comprises the sequence of SEQ ID NO: 658. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 666 and the sense strand comprises the sequence of SEQ ID NO: 659.
  • the antisense strand comprises the sequence of SEQ ID NO: 691 and the sense strand comprises the sequence of SEQ ID NO: 682. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 692 and the sense strand comprises the sequence of SEQ ID NO: 683. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 693 and the sense strand comprises the sequence of SEQ ID NO: 684. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 694 and the sense strand comprises the sequence of SEQ ID NO: 685.
  • the antisense strand comprises the sequence of SEQ ID NO: 695 and the sense strand comprises the sequence of SEQ ID NO: 686. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 696 and the sense strand comprises the sequence of SEQ ID NO: 687. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 697 and the sense strand comprises the sequence of SEQ ID NO: 688. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 698 and the sense strand comprises the sequence of SEQ ID NO: 689. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 699 and the sense strand comprises the sequence of SEQ ID NO: 690.
  • each of B 1 and B 2 is a nucleobase; and R 1 is selected from the group consisting of hydrogen and C 1-6 alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide.
  • the antisense strand has a 3’ end nucleotide overhang compared to the sense strand.
  • the 3’ end nucleotide overhang comprises 1. 2, 3, 4, 5, or 6 nucleotides compared to the sense strand.
  • the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand.
  • the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary.
  • the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides.
  • the antisense strand comprises a nucleotide sequence that is at least about 60%. 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of INHBE mRNA.
  • the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the INHBE mRNA.
  • the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the INHBE mRNA.n some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide.
  • the modified nucleotide is at least one of a modified nucleotide selected from the group consisting of: a 2'- O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2’- fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3’ linked nucleotide or 5’- 5’ linked nucleotide), a thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising a modified nucleotide component represented by Formula (I):
  • each of B 1 and B 2 is a nucleobase; and R 1 is selected from the group consisting of hydrogen and C 1-6 alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide, and a nucleotide comprising a modified nucleotide component represented by Formula (II).
  • each of B 1 and B 2 is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof.
  • each of B 1 and B 2 is independently selected from adenine, uracil, cytosine, and modified analogs thereof.
  • R 1 is C 1-6 alkyl. In some embodiments, wherein R 1 is -CH3. In some embodiments, B 1 is uracil. In some embodiments, R 1 is -CH3 and B 1 is uracil. In some embodiments, B 2 is adenine. In some embodiments, B 2 is uracil. In some embodiments, the sense strand comprises an inverted deoxyribonucleotide at the 5’ end; optionally wherein the inverted deoxyribonucleotide is a 5'-5' linked deoxythymidine.
  • the sense strand comprises an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide is a 3'-3’ linked deoxythymidine.
  • the sense strand comprises an inverted deoxyribonucleotide at the 5’ end and an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide at the 5’ end is a 5'-5' linked deoxythymidine and the inverted deoxyribonucleotide at the 3’ end is a 3'-3' linked deoxy thymidine.
  • the sense strand comprises a nucleotide comprising the modified nucleotide component represented by Formula (I) at the 3’ end; optionally wherein R 1 is -CH3 and B 1 is uracil.
  • the modified nucleotide is at least one of: 5 ’-vinyl phosphonate nucleotide, a 5 ’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-MOE (methoxyethyl)nucleotide, and/or a 2’-arabino fluoro (2’-araF) nucleotide.
  • the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O-phosphonate.
  • the modified nucleotide is at least one of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2’-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’- amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and/or a non-natural base comprising nucleotide.
  • the antisense strand and/or the sense strand comprises at least one intemucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalky Iphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage.
  • the antisense strand and/or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and/or the sense strand comprises at least one a phosphorothioate (PS) bond.
  • the dsRNA further comprises a ligand or targeting moiety. In some embodiments, the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the dsRNA.
  • the ligand or targeting moiety is conjugated to the 5’ end of the sense strand of the dsRNA. In some embodiments, ligand or targeting moiety is at least one N-Acetyl-Galactosamine (GalNAc). In some embodiments, the ligand or targeting moiety is represented by Formula (I):
  • each occurrence of T 1 and T 2 is independently selected from 5-membered heterocyclyl and alkylene; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; L A is absent or a linker; and n is an integer from 1 to 6.
  • each occurrence of T 1 and T 2 is independently selected from 5-membered heterocyclyl having at least one ring oxygen and C 1-6 alkylene.
  • the dsRNA is represented by Formula (I-B-A):
  • the dsRNA is represented by Formula (I-B):
  • the dsRNA is represented by Formula (I-B-I-A):
  • the dsRNA is represented by Formula (I-B-I) : Formula (I-B-I) or a pharmaceutically acceptable salt thereof.
  • the dsRNA is represented by Formula (I-B-II-A):
  • the dsRNA is represented by Formula (I-B-II):
  • the dsRNA is represented by Formula (I-B-III-A): Formula (I-B-III-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.
  • the dsRNA is represented by Formula (I-B-III):
  • the dsRNA is represented by Formula (I-B-IV-A):
  • the dsRNA is represented by Formula (I-B-IV):
  • L A is absent.
  • L A is a cleavable linker.
  • L A is a non- cleavable linker.
  • a 1 is a double-stranded RNA (dsRNA) molecule, wherein L A is attached to only one strand of the dsRNA.
  • dsRNA double-stranded RNA
  • the compound is represented by Formula (I- A):
  • Formula (I- A) or a pharmaceutically acceptable salt thereof is represented by Formula (I- A- A): Formula (I-A-A) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (I-A-I):
  • Formula (I-A-I) or a pharmaceutically acceptable salt thereof is represented by Formula (I-A-I- A): Formula (I-A-I-A) or a pharmaceutically acceptable salt thereof.
  • the compound is represented by Formula (I- A- II):
  • the compound is represented by Formula (I-A-II- A): Formula (I-A-II-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.
  • the ligand or targeting moiety is tri-GalNAc6.
  • the ligand or targeting moiety is L96.
  • the ligand or targeting moiety is:
  • the ligand or targeting moiety is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • a cell comprising a dsRNA of the disclosure is provided.
  • a vector encoding at least one unmodified strand a dsRNA of the disclosure is provided, optionally both strands, of the disclosure is provided a cell comprising the vector is provided.
  • a pharmaceutical composition for inhibiting expression of INHBE comprising the dsRNA and a pharmaceutically acceptable carier, diluent, excipient, or combination thereof of the disclosure is provided.
  • a method of inhibiting INHBE expression in a cell comprising (a) contacting the cell with the dsRNA of the disclosure or the pharmaceutical composition of the disclosure; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an INHBE gene, thereby inhibiting expression of the INHBE gene in the cell, optionally wherein the method is in vivo.
  • the INHBE expression is inhibited by at least 30% relative to a control.
  • a method of treating a disorder mediated by or associated with INHBE comprising administering to a subject in need of such treatment a therapeutically effective amount of a dsRNA of the disclosure, or a pharmaceutical composition of the disclosure.
  • the disorder is a cardiovascular disorder.
  • the disorder is cardiovascular disease.
  • FIGs. 1A-C show bar graphs of the percent (%) inhibition of INHBE mRNA in Huh- 7 cells transfected with the indicated GalNAc conjugated, modified siRNA at lOnM, and 0. InM, relative to INHBE mRNA in mock-treated cells. INHBE mRNA level measured by quantitative PCR and normalized to GAPDH.
  • FIG. 2 shows graphs of exemplary INHBE siRNA compounds in Huh7 cell line in a single dose screen at 100nM, 33nM, 1 InM, 3.7nM, 1.2nM, 0.412nM, 0.137nM, and 0.046nM of the selected siRNA.
  • INHBE mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells and the average KD and SD was determined.
  • FIG. 3 shows a bar graph of the percent (%) knockdown of INHBE mRNA in human hepatocytes cells treated with indicated GalNAc conjugated, modified siRNA at lOnM, and InM relative to INHBE mRNA in PBS treated cells. INHBE mRNA level measured by quantitative PCR and normalized to GAPDH.
  • FIG. 4 shows a graph of the relative expression of human INHBE mRNA in hydrodynamic injection model with the indicated 13 conjugated, modified siRNA at 1 mg/kg, relative to INHBE expression in PBS treated mice. INHBE mRNA levels were measured by quantitative PCR and normalized to NEO.
  • FIG. 5 shows a graph of the relative expression of human INHBE mRNA in hydrodynamic injection model with the indicated 12 conjugated, modified siRNA at 1 mg/kg or 1.5 mg/kg, relative to INHBE expression in PBS treated mice.
  • INHBE mRNA levels were measured by quantitative PCR and normalized to NEO.
  • FIG. 6 shows a bar graph of the relative expression of INHBE mRNA in a non-human primate model treated with siRNA Compounds 100639 and 100642 at 5 mg/kg. INHBE mRNA levels were measured via quantitative PCR using liver biopsy samples, and normalized to INHBE expression level at day -4 for each individual animal.
  • FIGs. 7A-7C show graphs depicting the agonistic activity of tested compounds in a cell-based hTLR7 reporter assay (FIG. 7A), hTLR8 reporter assay (FIG. 7B), and hTLR9 reporter assay (FIG. 7C).
  • the y-axis shows the level of activity as a fold change over unstimulated cells.
  • the x-axis shows the concentration of each compound in nM (loglO scale).
  • FIG. 8 shows a volcano plot of differentially expresses genes (DEGs) among different groups in primary human hepatocyte (PHH) cells treated with indicated exemplary siRNA compounds.
  • the x-axis represents the log 2 (FoldChange), while y-axis represents statistical significance for each gene.
  • FIG. 9 shows graphs depicting the biochemical tests of mice over 7 days after receipt of a single dose of PBS control or siRNA compound 100635, 100642, or 100643.
  • the mean plasma concentration of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TRIG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), creatinine (CREZ), cholesterol (CHOL), lactate dehydrogenase (LDH), urine micro total protein (UP), and plasma urea (UREA) is shown.
  • FIG. 10 provides a scheme of the non-human primate study of Example 10.
  • FIG. 11 shows knockdown of INHBE expression in the liver of the non-human primate subjects described in Example 10.
  • FIG. 12 provides a scheme of the non-human primate study of Example 11.
  • FIG. 13 shows knockdown of INHBE expression in the liver of the non-human primate subjects described in Example 11.
  • FIGs. 14A-14D show the pharmacodynamics of Compound 100639 in spontaneously obese Cynomolgus monkeys.
  • FIG 14A shows the percentage of body weight change in Cynomolgus monkeys from baseline over time .
  • FIG 14B shows the BMI change in Cynomolgus monkeys from baseline over time.
  • FIG 14C shows the relative level of INHBE gene expression in the liver of Cynomolgus monkeys as compared to baseline over time.
  • FIG 14D shows the relative percentage of INHBE protein in plasma of Cynomolgus monkeys as compared to baseline over time.
  • FIG. 15 provides concentrations and conditions of siRNA compounds used in the DRC study of Example 12.
  • FIGs. 16A-16J show percent inhibition INHBE dose-response plots of exemplary siRNA compounds of Table 13.
  • FIG. 17 shows a graph of the relative expression of human INHBE mRNA in humanized INHBE transgenic mice with the indicated 4 conjugated, modified siRNA at 3 mg/kg, relative to INHBE expression in PBS treated mice.
  • INHBE mRNA levels were measured by RT-qPCR (reverse transcription quantitative real-time polymerase chain reaction).
  • the disclosure provides dsRNA oligonucleotides and methods of using the dsRNA oligonucleotides for inhibiting the expression of a Lipoprotein(A) (INHBE) gene in a cell or a mammal where the dsRNA oligonucleotide targets a INHBE gene.
  • the disclosure also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of a INHBE gene, e.g., cardiovascular disease.
  • An INHBE dsRNA oligonucleotide directs the sequence-specific degradation of INHBE mRNA.
  • reference to a range of 1-5,000-fold comprises 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold, etc., as well as 1.1-, 1.2-, 1.3-, 1.4-, or 1.5-fold, etc., 2.1-, 2.2-, 2.3-, 2.4-, or 2.5-fold, etc., and so forth.
  • an element means one element or more than one element, e.g., a plurality of elements.
  • the term “at least” 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.
  • the number of nucleotides in a nucleic acid molecule must be an integer.
  • “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property.
  • nucleotide overhang As used herein, “no more than” or “less than” 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.
  • G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively.
  • T and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine.
  • ribonucleotide or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety.
  • guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine may he replaced in the nucleotide sequences of the disclosure by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the disclosure.
  • INHBE' refers to the Inhibin Subunit Beta E gene. According to the NCBI NLM website, this gene encodes his gene encodes a member of the TGF-beta (transforming growth factor-beta) superfamily of proteins. The encoded preproprotein is proteolytically processed to generate an inhibin beta subunit. Inhibins have been implicated in regulating numerous cellular processes including cell proliferation, apoptosis, immune response and hormone secretion. This gene may be upregulated under conditions of endoplasmic reticulum stress, and this protein may inhibit cellular proliferation and growth in pancreas and liver..
  • a human 1NHBE mRNA sequence is GenBank accession number NM_031479.5, included herein as SEQ ID NO: 588.
  • a rhesus monkey (Macaca mulatto) INHBE mRNA sequence is GenBank accession number XM_028847001.1.
  • target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a INHBE gene, including mRNA that is a product of RNA processing of a primary transcription product.
  • 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.
  • 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.
  • a first nucleotide sequence can be described as complementary to a second nucleotide sequence when the two sequences hybridize (e.g., anneal) under stringent hybridization conditions.
  • Hybridization conditions include temperature, ionic strength, pH, and organic solvent concentration for the annealing and/or washing steps.
  • stringent hybridization conditions refers to conditions under which a first nucleotide sequence will hybridize preferentially to its target sequence, e.g., a second nucleotide sequence, and to a lesser extent to, or not at all to, other sequences.
  • Stringent hybridization conditions are sequence dependent, and are different under different environmental parameters.
  • stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the nucleotide sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the first nucleotide sequences hybridize to a perfectly matched target sequence.
  • sequences can be referred to as “fully complementary” with respect to each other herein.
  • first sequence is referred to as “substantially complementary” with respect to a second sequence herein
  • the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3, or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application.
  • 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, may yet be referred to as “fully complementary” for the purposes described herein.
  • “Complementary” sequences may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
  • non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogsteen base pairing.
  • 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 INHBE) including a 5’ UTR, an open reading frame (ORF), or a 3’ UTR.
  • mRNA messenger RNA
  • a polynucleotide is complementary to at least a part of an INHBE mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding INHBE.
  • the antisense strand of the dsRNA is sufficiently complementary to a target mRNA so as to cause cleavage of the target mRNA.
  • double-stranded RNA refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands.
  • dsRNA double-stranded RNA
  • the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide.
  • dsRNA may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA’' for the purposes of this specification and claims.
  • 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.’ 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.
  • a dsRNA may comprise one or more nucleotide overhangs.
  • siRNA is also used herein to refer to a dsRNA as described above.
  • nucleotide overhang refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3’-end of one strand of the dsRNA extends beyond the 5’-end of the other strand, or vice versa.
  • “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, /. ⁇ ?., no nucleotide overhang.
  • a “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
  • antisense strand refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence.
  • region of complementarity refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5’ and/or 3’ terminus.
  • sense strand refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
  • “Introducing into a cell,” when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro', a dsRNA may also be "introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism.
  • dsRNA 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 or known in the art.
  • the terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of’ and the like in as far as they refer to a INHBE gene herein refer to the at least partial suppression of the expression of a INHBE gene, as manifested by a reduction of the amount of mRNA which may be isolated from a first cell or group of cells in which a INHBE gene is transcribed and which has or have been treated such that the expression of a INHBE 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 or have not been so treated (control cells).
  • the degree of inhibition is usually expressed in terms of
  • the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to INHBE gene expression, e.g., the amount of protein encoded by an INHBE gene which is secreted by a cell, the level of plasma lipid levels or the number of cells displaying a certain phenotype.
  • INHBE gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay.
  • the assays provided in the Examples below shall serve as such reference.
  • expression of a INHBE gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide of the disclosure.
  • a INHBE gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the disclosure.
  • a INHBE gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded of the disclosure.
  • the terms “treat,” “treatment,” and the like refer to relief from or alleviation of pathological processes mediated by INHBE expression.
  • the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
  • the phrases “effective amount” refers to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by INHBE expression or an overt symptom of pathological processes mediated by INHBE expression.
  • the specific amount that is effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by INHBE expression, the patient’s history and age, the stage of pathological processes mediated by INHBE expression, and the administration of other anti-pathological processes mediated by INHBE expression agents.
  • a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier.
  • pharmaceutically effective amount refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
  • a therapeutically effective amount of a dsRNA targeting INHBE can reduce INHBE serum levels by at least 25%.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the term specifically excludes cell culture medium.
  • pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • tri-GalNAc6 refers to the structure:
  • L96 ligand or “L96” refers to the structure: IL Double-stranded Ribonucleic Acids (dsRNA)
  • dsRNA double-stranded ribonucleic acid
  • INHBE gene double-stranded ribonucleic acid
  • the dsRNA comprises an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA or an mRNA fragment formed in the expression of a INHBE gene. In some embodiments, the dsRNA comprises at least 70% complementarity to the mRNA or the fragment mRNA of human INHBE mRNA.
  • the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 598, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 589; (b) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 599, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 590; (c) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 600, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO
  • the disclosure provides for a double- stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE, wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 607; (b) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 617, and sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 608; (c) the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 618, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID
  • dsRNA double- stranded ribonucleic acid
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length
  • the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 607
  • the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 617, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 608
  • the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 618, and the sense strand a sequence that is at least 70% or 80% identical
  • the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE comprising a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: (a) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 598, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589; (b) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 599, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 590; (c) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence
  • the disclosure provides for a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of INHBE, wherein the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein: the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 616, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 607; (b) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence comprising the sequence of SEQ ID NO: 617, and the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 608; (c) the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence
  • the INHBE gene is human INHBE. In some embodiments, the INHBE is human INHBE comprising the sequence shown in SEQ ID NO: 588 (NM_031479.5).
  • the sense strand is 70%, 80%, 90%, 95% or more identical to the sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 6
  • the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 589
  • the sense strand comprises: (a) 20 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 6
  • the antisense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 624, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO:
  • the antisense strand comprises: (a) 21 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO:
  • SEQ ID NO: 697 SEQ ID NO: 698, or SEQ ID NO: 699;
  • 22 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 624, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO
  • the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 6
  • the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO:
  • the antisense strand is selected from an antisense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO:
  • the antisense strand comprises the sequence of SEQ ID NO: 598 and the sense strand comprises the sequence of SEQ ID NO: 589. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 599 and the sense strand comprises the sequence of SEQ ID NO: 590. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 600 and the sense strand comprises the sequence of SEQ ID NO: 591. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 601 and the sense strand comprises the sequence of SEQ ID NO: 592.
  • the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 593. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 700. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 603 and the sense strand comprises the sequence of SEQ ID NO: 594. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 604 and the sense strand comprises the sequence of SEQ ID NO: 595.
  • the antisense strand comprises the sequence of SEQ ID NO: 605 and the sense strand comprises the sequence of SEQ ID NO: 596. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 606 and the sense strand comprises the sequence of SEQ ID NO: 597. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 616 and the sense strand comprises the sequence of SEQ ID NO: 607. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 617 and the sense strand comprises the sequence of SEQ ID NO: 608. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 618 and the sense strand comprises the sequence of SEQ ID NO: 609.
  • the antisense strand comprises the sequence of SEQ ID NO: 619 and the sense strand comprises the sequence of SEQ ID NO: 610. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 620 and the sense strand comprises the sequence of SEQ ID NO: 611. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 621 and the sense strand comprises the sequence of SEQ ID NO: 612. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 622 and the sense strand comprises the sequence of SEQ ID NO: 613.
  • the antisense strand comprises the sequence of SEQ ID NO: 623 and the sense strand comprises the sequence of SEQ ID NO: 614. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 624 and the sense strand comprises the sequence of SEQ ID NO: 615. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 665 and the sense strand comprises the sequence of SEQ ID NO: 658. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 666 and the sense strand comprises the sequence of SEQ ID NO: 659.
  • the antisense strand comprises the sequence of SEQ ID NO: 691 and the sense strand comprises the sequence of SEQ ID NO: 682. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 692 and the sense strand comprises the sequence of SEQ ID NO: 683. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 693 and the sense strand comprises the sequence of SEQ ID NO: 684. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 694 and the sense strand comprises the sequence of SEQ ID NO: 685.
  • the antisense strand comprises the sequence of SEQ ID NO: 695 and the sense strand comprises the sequence of SEQ ID NO: 686. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 696 and the sense strand comprises the sequence of SEQ ID NO: 687. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 697 and the sense strand comprises the sequence of SEQ ID NO: 688. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 698 and the sense strand comprises the sequence of SEQ ID NO: 689. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 699 and the sense strand comprises the sequence of SEQ ID NO: 690.
  • the sense strand is 70%, 80%, 90%, 95% or more identical to the sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594,
  • SEQ ID NO: 614 SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682,
  • SEQ ID NO: 683 SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687,
  • the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 6
  • the sense strand comprises: (a) 20 contiguous nucleotides of a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596,
  • SEQ ID NO: 658 SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684,
  • the antisense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ
  • the antisense strand comprises: (a) 21 contiguous nucleotides of an antisense sense strand sequence comprising the sequence of SEQ ID NO:
  • SEQ ID NO: 600 SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO:
  • SEQ ID NO: 618 SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 623, SEQ ID NO: 624, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, or SEQ ID NO: 699.
  • the sense strand sequence is selected from a sense strand sequence comprising the sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 61 1 , SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, or SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO:
  • the sense strand sequence is selected from a sense strand sequence of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 700, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, ID NO: 597, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608, SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, or SEQ ID NO: 615, SEQ ID NO: 658, SEQ ID NO: 659, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ ID NO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689
  • the antisense strand is selected from an antisense strand sequence of SEQ ID NO: 598, SEQ ID NO: 599, SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ ID NO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 616, SEQ ID NO: 617, SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ ID NO: 622, SEQ ID NO: 623, or SEQ ID NO: 624, SEQ ID NO: 665, SEQ ID NO: 666, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ ID NO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698, or SEQ ID NO: 699.
  • the antisense strand comprises the sequence of SEQ ID NO:
  • the antisense strand comprises the sequence of SEQ ID NO: 599 and the sense strand comprises the sequence of SEQ ID NO: 590.
  • the antisense strand comprises the sequence of SEQ ID NO: 600 and the sense strand comprises the sequence of SEQ ID NO: 591.
  • the antisense strand comprises the sequence of SEQ ID NO: 601 and the sense strand comprises the sequence of SEQ ID NO: 592.
  • the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 593.
  • the antisense strand comprises the sequence of SEQ ID NO: 602 and the sense strand comprises the sequence of SEQ ID NO: 700. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 603 and the sense strand comprises the sequence of SEQ ID NO: 594. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 604 and the sense strand comprises the sequence of SEQ ID NO: 595. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 605 and the sense strand comprises the sequence of SEQ ID NO: 596.
  • the antisense strand comprises the sequence of SEQ ID NO: 606 and the sense strand comprises the sequence of SEQ ID NO: 597. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 616 and the sense strand comprises the sequence of SEQ ID NO: 607. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 617 and the sense strand comprises the sequence of SEQ ID NO: 608. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 618 and the sense strand comprises the sequence of SEQ ID NO: 609. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 619 and the sense strand comprises the sequence of SEQ ID NO: 610.
  • the antisense strand comprises the sequence of SEQ ID NO: 620 and the sense strand comprises the sequence of SEQ ID NO: 611. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 621 and the sense strand comprises the sequence of SEQ ID NO: 612. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 622 and the sense strand comprises the sequence of SEQ ID NO: 613. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 623 and the sense strand comprises the sequence of SEQ ID NO: 614.
  • the antisense strand comprises the sequence of SEQ ID NO: 624 and the sense strand comprises the sequence of SEQ ID NO: 615. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 665 and the sense strand comprises the sequence of SEQ ID NO: 658. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 666 and the sense strand comprises the sequence of SEQ ID NO: 659. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 691 and the sense strand comprises the sequence of SEQ ID NO: 682.
  • the antisense strand comprises the sequence of SEQ ID NO: 692 and the sense strand comprises the sequence of SEQ ID NO: 683. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 693 and the sense strand comprises the sequence of SEQ ID NO: 684. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 694 and the sense strand comprises the sequence of SEQ ID NO: 685. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 695 and the sense strand comprises the sequence of SEQ ID NO: 686.
  • the antisense strand comprises the sequence of SEQ ID NO: 696 and the sense strand comprises the sequence of SEQ ID NO: 687. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 697 and the sense strand comprises the sequence of SEQ ID NO: 688. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 698 and the sense strand comprises the sequence of SEQ ID NO: 689. In some embodiments, the antisense strand comprises the sequence of SEQ ID NO: 699 and the sense strand comprises the sequence of SEQ ID NO: 690.
  • At least one nucleotide of the dsRNA is a modified nucleotide selected from the group consisting of: a 5 ’-vinyl phosphonate nucleotide, a 2'-O-methyl modified nucleotide, an inverted deoxyribonucleotide (3'-3' linked nucleotide or 5 ’-5’ linked nucleotide), a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide, a nucleotide comprising a modified nucleotide component represented by Formula (I):
  • each of B 1 and B 2 is a nucleobase; and R 1 is selected from the group consisting of hydrogen and C 1-6 alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide.
  • the antisense strand has a 3’ end nucleotide- overhang compared to the sense strand.
  • the 3’ end nucleotide overhang comprises 1, 2, 3, 4, 5. or 6 nucleotides compared to the sense strand.
  • the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand.
  • the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary.
  • the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides.
  • the antisense strand comprises a nucleotide sequence that is at. least about 60%. 65%, 70%, 75%. 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of INHBE mRNA.
  • the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the INHBE mRNA.
  • the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the INHBE mRNA.n some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide.
  • the modified nucleotide is at least one of a modified nucleotide selected from the group consisting of: a 2'- O-methyl modified nucleotide, a nucleotide comprising a 5’-phosphorothioate group, a 2’- fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide or 5’- 5’ linked nucleotide), a thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising a modified nucleotide component represented by Formula (I):
  • each of B 1 and B 2 is a nucleobase; and R 1 is selected from the group consisting of hydrogen and C 1-6 alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide, and a nucleotide comprising a modified nucleotide component represented by Formula (II).
  • each of B 1 and B 2 is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof.
  • each of B 1 and B 2 is independently selected from adenine, uracil, cytosine, and modified analogs thereof.
  • R 1 is C 1-6 alkyl. In some embodiments, wherein R 1 is -CH3. In some embodiments, B 1 is uracil. In some embodiments, R 1 is -CH3 and B 1 is uracil. In some embodiments, B 2 is adenine. In some embodiments, B 2 is uracil. In some embodiments, the sense strand comprises an inverted deoxyribonucleotide at the 5’ end; optionally wherein the inverted deoxyribonucleotide is a 5'-5' linked deoxy thymidine.
  • the sense strand comprises an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide is a 3'-3’ linked deoxythymidine.
  • the sense strand comprises an inverted deoxyribonucleotide at the 5’ end and an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide at the 5’ end is a 5'-5' linked deoxythymidine and the inverted deoxyribonucleotide at the 3’ end is a 3'-3' linked deoxy thymidine.
  • the sense strand comprises a nucleotide comprising the modified nucleotide component represented by Formula (I) at the 3’ end; optionally wherein R 1 is -CH3 and B 1 is uracil.
  • the modified nucleotide is at least one of: 5 ’-vinyl phosphonate nucleotide, a 5 ’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-M0E (methoxyethyl)nucleotide, and/or a 2’-arabino fluoro (2’-araF) nucleotide.
  • the antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl-phosphonate or a 4'-O-phosphonate.
  • the modified nucleotide is at least one of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2’-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’- amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and/or a non-natural base comprising nucleotide.
  • the antisense strand and/or the sense strand comprises at least one intemucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage.
  • the antisense strand and/or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and/or the sense strand comprises at least one a phosphorothioate (PS) bond.
  • PS phosphorothioate
  • the dsRNA further comprises a ligand or targeting moiety.
  • the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA.
  • the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the dsRNA.
  • the ligand or targeting moiety is conjugated to the 5’ end of the sense strand of the dsRNA.
  • ligand or targeting moiety is at least one N-Acetyl-Galactosamine (GalNAc).
  • the ligand or targeting moiety is represented by represented by Formula (I):
  • each occurrence of T 1 and T 2 is independently selected from 5-membered heterocyclyl and alkylene; each occurrence of X is selected from the group consisting of -OH and -SH; and each occurrence of L is a linker; L A is absent or a linker; and n is an integer from 1 to 6.
  • each occurrence of T 1 and T 2 is independently selected from 5-membered heterocyclyl having at least one ring oxygen and C 1-6 alkylene.
  • the dsRNA is represented by Formula (I-B-A):
  • the dsRNA is represented by Formula (I-B):
  • the dsRNA is represented by Formula (I-B-I-A):
  • the dsRNA is represented by Formula (I-B-I) : Formula (I-B-I) or a pharmaceutically acceptable salt thereof.
  • the dsRNA is represented by Formula (I-B-II-A):
  • the dsRNA is represented by Formula (I-B-II):
  • the dsRNA is represented by Formula (I-B-III-A): Formula (I-B-III-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of g and h is an integer from 1-20.
  • the dsRNA is represented by Formula (I-B-III):
  • the dsRNA is represented by Formula (I-B-IV-A):
  • the dsRNA is represented by Formula (I-B-IV):
  • L A is absent.
  • L A is a cleavable linker.
  • L A is a non- cleavable linker.
  • a 1 is a double-stranded RNA (dsRNA) molecule, wherein L A is attached to only one strand of the dsRNA.
  • dsRNA double-stranded RNA
  • the compound is represented by Formula (I- A):
  • Formula (I- A) or a pharmaceutically acceptable salt thereof is represented by Formula (I- A- A): Formula (I-A-A) or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by Formula (I-A-I):
  • Formula (I-A-I) or a pharmaceutically acceptable salt thereof is represented by Formula (I-A-I- A): Formula (I-A-I-A) or a pharmaceutically acceptable salt thereof.
  • the compound is represented by Formula (I- A- II):
  • the compound is represented by Formula (I-A-II- A): Formula (I-A-II-A) or a pharmaceutically acceptable salt thereof, wherein each occurrence of a and b is an integer from 1-20.
  • the ligand or targeting moiety is tri-GalNAc6.
  • the ligand or targeting moiety is L96.
  • the ligand or targeting moiety is:
  • the ligand or targeting moiety is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence shown in Table 1 and Table 2.
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises a sequence that is at least 70% or 80% identical to an antisense strand sequence shown in Table 1 and Table 2.
  • the dsRNA is Compound 100494, 100506, 100509, 100535, 100557, 100561, 100563, 100563, 100569, 100580, 100589, 100604, 100613, 100625, and 100629.
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence shown in Table 6 and Table 7.
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises a sequence that is at least 70% or 80% identical to an antisense strand sequence shown in Table 6 and Table 7.
  • the dsRNA is selected from the group consisting of Compound 100635, Compound 100636, Compound 100637, Compound 100638, Compound 100639, Compound 100640, Compound 100641, Compound 100642, Compound 100643, Compound 100644, Compound 100645, Compound 100646, and Compound 100647.
  • the dsRNA is Compound 100635. In some embodiments, the dsRNA is Compound 100636. In some embodiments, the dsRNA is Compound 100637. In some embodiments, the dsRNA is Compound 100638. In some embodiments, the dsRNA is Compound 100639. In some embodiments, the dsRNA is Compound 100640. In some embodiments, the dsRNA is Compound 100641. In some embodiments, the dsRNA is Compound 100642. In some embodiments, the dsRNA is Compound 100643. In some embodiments, the dsRNA is Compound 100644. In some embodiments, the dsRNA is Compound 100645. In some embodiments, the dsRNA is Compound 100646. In some embodiments, the dsRNA is Compound 100647.
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence shown in Table 9 and Table 10.
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises a sequence that is at least 70% or 80% identical to an antisense strand sequence shown in Table 9 and Table 10.
  • the dsRNA is selected from the group consisting of Compound 100643, Compound 100647, Compound 100648, Compound 100649, Compound 100650, Compound 100651, Compound 100652, 100653, Compound 100654, Compound 100655, Compound 100656, and Compound 100657.
  • the dsRNA is Compound 100647. In some embodiments, the dsRNA is Compound 100648. In some embodiments, the dsRNA is Compound 100649. In some embodiments, the dsRNA is Compound 100650. In some embodiments, the dsRNA is Compound 100651. In some embodiments, the dsRNA is Compound 100652. In some embodiments, the dsRNA is Compound 100653. In some embodiments, the dsRNA is Compound 100654. In some embodiments, the dsRNA is Compound 100655. In some embodiments, the dsRNA is Compound 100656. In some embodiments, the dsRNA is Compound 100657.
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense strand sequence shown in Table 13.
  • the dsRNA comprises a sense strand and an antisense strand each 15 to 30 nucleotides in length, wherein the antisense strand comprises a sequence that is at least 70% or 80% identical to an antisense strand sequence shown in Table 13.
  • the dsRNA is selected from the group consisting of Compound 100643, Compound 100658, Compound 100659, Compound 100660, Compound 100661 , Compound 100662, Compound 100663, Compound 100664, Compound 100665, and Compound 100666.
  • the dsRNA is Compound 100658. In some embodiments, the dsRNA is Compound 100659. In some embodiments, the dsRNA is Compound 100660. In some embodiments, the dsRNA is Compound 100661. In some embodiments, the dsRNA is Compound 100662. In some embodiments, the dsRNA is Compound 100663. In some embodiments, the dsRNA is Compound 100664. In some embodiments, the dsRNA is Compound 100665. In some embodiments, the dsRNA is Compound 100666.
  • the INHBE is human INHBE. In some embodiments, the INHBE is human INEIBE comprising the sequence shown in SEQ ID NO: 588 (NM_031479.5).
  • the sense strand is 70%, 80%, 90%, 95%, or more identical to the sense strands listed in Table 1 and Table 2. In some embodiments, the sense strand is 70%, 80%, 90%, 95%, or more identical to the sense strands listed in Table 6 and Table 7. In some embodiments, the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence shown in Table 6 and Table 7. In some embodiments, the sense strand is 70%, 80%, 90%, 95%, or more identical to the sense strands listed in Table 9 and Table 10. In some embodiments, the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence shown in Table 9 and Table 10.
  • the sense strand comprises at least 15 contiguous nucleotides of a sense strand sequence shown in Table 1 and Table 2. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of a sense strand sequence shown in Table 1 and Table 2. In some embodiments, the sense strand comprises 21 contiguous nucleotides of a sense strand sequence shown in Table 1 and Table 2. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in Table 1 and Table 2. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a sense strand sequence shown in Table 6 and Table 7.
  • the sense strand comprises 21 contiguous nucleotides of a sense strand sequence shown in Table 6 and Table 7. In some embodiments, the sense strand comprises 22 contiguous nucleotides of a sense strand sequence shown in Table 6 and Table 7. In some embodiments, the sense strand comprises 23 contiguous nucleotides of a sense strand sequence shown in Table 6 and Table 7. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in Table 6 and Table 7. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of a sense strand sequence shown in Table 9 and Table 10.
  • the sense strand comprises 21 contiguous nucleotides of a sense strand sequence shown in Table 9 and Table 10. In some embodiments, the sense strand comprises 22 contiguous nucleotides of a sense strand sequence shown in Table 9 and Table 10. In some embodiments, the sense strand comprises 23 contiguous nucleotides of a sense strand sequence shown in Table 9 and Table 10. In some embodiments, the sense strand sequence is selected from a sense strand sequence shown in Table 9 and Table 10.
  • the antisense strand is 70%, 80%, 90%, 95%, or more identical to the antisense strands listed in Table 1 and Table 2.
  • the antisense strand comprises at least 16, 17, 18, 19, 20, or 21 contiguous nucleotides of an antisense sense strand sequence shown in Table 1 and Table 2.
  • the antisense strand comprises 21 contiguous nucleotides of an antisense sense strand sequence shown in Table 1 and Table 2.
  • the antisense strand is selected from an antisense strand sequence shown in Table 1 and Table 2.
  • the antisense strand is 70%, 80%, 90%, 95%, or more identical to the antisense strands listed in Table 6 and Table 7. In some embodiments, the antisense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides of an antisense sense strand sequence shown in Table 6 and Table 7. In some embodiments, the antisense strand comprises 21 contiguous nucleotides of an antisense sense strand sequence shown Table 6 and Table 7. In some embodiments, the antisense strand comprises 22 contiguous nucleotides of an antisense sense strand sequence shown Table 6 and Table 7.
  • the antisense strand comprises 23 contiguous nucleotides of an antisense sense strand sequence shown Table 6 and Table 7. In some embodiments, the antisense strand is selected from an antisense strand sequence shown in Table 6 and Table 7. In some embodiments, the antisense strand is 70%, 80%, 90%, 95%, or more identical to the antisense strands listed in Table 9 and Table 10. In some embodiments, the antisense strand comprises at least 16, 17, 18, 19, 20, 21 , 22, or 23 contiguous nucleotides of an antisense sense strand sequence shown in Table 9 and Table 10. In some embodiments, the antisense strand comprises 21 contiguous nucleotides of an antisense sense strand sequence shown Table 9 and Table 10.
  • the antisense strand comprises 22 contiguous nucleotides of an antisense sense strand sequence shown Table 9 and Table 10. In some embodiments, the antisense strand comprises 23 contiguous nucleotides of an antisense sense strand sequence shown Table 9 and Table 10. In some embodiments, the antisense strand is selected from an antisense strand sequence shown in Table 9 and Table 10.
  • the sense strand sequence is selected from a sense strand sequence shown in Table 1 and Table 2, and the antisense strand is selected from an antisense strand sequence shown in Table 1 and Table 2.
  • the sense strand sequence is selected from a sense strand sequence shown in Table 6 and Table 7, and the antisense strand is selected from an antisense strand sequence shown in Table 6 and Table 7.
  • the sense strand sequence is selected from a sense strand sequence shown in Table 9 and Table 10
  • the antisense strand is selected from an antisense strand sequence shown in Table 9 and Table 10.
  • the sense strand sequence is selected from a sense strand sequence shown in Table 13 and Table 14, and the antisense strand is selected from an antisense strand sequence shown in Table 13 and Table 14.
  • the dsRNA has a mismatch to a fragment of INHBE mRNA. In some embodiments, the dsRNA comprises one or two mismatches to the mRNA or fragment of human INHBE mRNA. In some embodiments, the dsRNA is more than 70% identical to the mRNA or fragment of human INHBE mRNA. In some embodiments, the dsRNA is more than 70%, 75%, 80%, 85%, 90%, or 95 % identical to the mRNA or fragment of human INHBE mRNA.
  • the antisense strand comprises a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a target mRNA corresponding to a fragment of INHBE mRNA.
  • the antisense strand of the dsRNA comprises at least 80% complementarity to the fragment of the INHBE mRNA.
  • the mismatch is in the sense strand.
  • the mismatch is in the antisense strand.
  • the antisense strand of the dsRNA comprises one, two, three, or four mismatches to the fragment of the INHBE mRNA.
  • the mismatch is located in the middle of the dsRNA. In some embodiments, the mismatch is in the 5’ or 3’ region of the dsRNA. In some embodiments, the mismatch is no more than 5 nucleotides from the 5’ or 3’ end of the dsRNA.
  • At least one strand of the dsRNA comprises a 3’ or 5’ overhang of at least 1 nucleotide.
  • the overhang is at least 2 or a at least 3 nucleotides.
  • the overhang is at least 4, at least 5, or at least 6 nucleotides.
  • in the dsRNA at least one strand comprises a 3’ overhang.
  • in the dsRNA at least one strand comprises a 5’ overhang.
  • the antisense strand has a 3’ end nucleotide overhang compared to the sense strand.
  • the 3’ end nucleotide overhang comprises 1, 2, 3, 4. 5, or 6 nucleotides compared to die sense strand. In some embodiments, the 3’ end nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. In some embodiments, the antisense strand and the sense strand are at least 80% complementary. In some embodiments, the antisense strand and the sense strand comprise at least one, at least two, at least three, or at least four mismatched nucleotides.
  • the dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
  • the dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure.
  • the duplex structure is between 15 and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length.
  • the duplex is 19 base pairs in length.
  • the duplex is 20 base pairs in length.
  • the duplex is 21 base pairs in length.
  • each strand of the dsRNA of the disclosure is between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is about 25-30 nucleotides in length. In some embodiments, each strand of the duplex is the same length or of different lengths. When two different ssRNAs are used in combination, the lengths of each strand of each ssRNA can be identical or can differ.
  • the dsRNA includes dsRNA that is longer than 21-23 nucleotides, e.g., dsRNA that is long enough to be processed by the RNase III enzyme Dicer into 21-23 base pair siRNA which is then incorporated into a RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • a dsRNA of the disclosure is at least 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or at least 100 base pairs in length.
  • Inhibition of the expression of the INHBE gene can be assayed by, for example, a nucleic acid based assay, such as by quantitative PCR, or by a protein-based method, such as by Western blot. Expression of a INHBE gene can be reduced by at least 50% when measured by an assay as described in the Examples below.
  • expression of a INHBE gene in cell culture, such as in Huh-7 cells can be assayed by measuring INHBE mRNA levels, such as by quantitative PCR assay, or by measuring protein levels, such as by ELISA assay.
  • the disclosure provides a single- stranded antisense oligonucleotide RNAi.
  • An antisense oligonucleotide is a single- stranded oligonucleotide that is complementary to a sequence within the target mRNA.
  • 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.
  • Antisense oligonucleotides can also inhibit target protein expression by binding to the mRNA target and promoting mRNA target destruction via Rnase-H.
  • the single-stranded antisense RNA molecule can be about 13 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence.
  • the single-stranded antisense RNA molecule can comprise a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the antisense sequences in Table 1 and Table 2, Table 6 and Table 7, or Table 9 and Table 10.
  • the dsRNA is chemically modified to enhance stability of the dsRNA.
  • the nucleic acids featured in the disclosure may be synthesized and/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. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Specific examples of dsRNA compounds useful in this disclosure include dsRNAs containing modified backbones or non natural internucleoside linkages.
  • dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • a modified dsRNA backbone includes at least one of: a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide; an inverted abasic nucleotide, a thymidine-glycol nucleic acid (GNA) S-Isomer; an inosine, and inverted deoxyribonucleotide (3'-3' linked nucleotide or 5’ -5 ’ linked nucleotide), a thymidine-glycol nucleic acid (GNA) S-Isomer, a nucleotide comprising a modified nucleotide component represented by Formula (I):
  • R 1 is selected from the group consisting of hydrogen and C 1-6 alkyl; optionally wherein the antisense strand and the sense strand each comprise at least one modified nucleotide.
  • the nucleotide comprising a modified nucleotide component represented by Formula (I) comprises a nucleobase represented by B 1 , wherein B 1 is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof.
  • the nucleotide comprising a modified nucleotide component represented by Formula (II) comprises a nucleobase represented by B 2 , wherein B 2 is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof.
  • each of B 1 and B 2 is independently selected from adenine, uracil, cytosine, and modified analogs thereof.
  • R 1 is C 1-6 alkyl.
  • R 1 is -CH3.
  • B 1 is uracil.
  • R 1 is -CH3 and B 1 is uracil.
  • B 2 is adenine.
  • B 2 is uracil.
  • the sense strand comprises an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide is a 3'-3' linked deoxythymidine.
  • the sense strand comprises an inverted deoxyribonucleotide at the 5’ end and an inverted deoxyribonucleotide at the 3’ end; optionally wherein the inverted deoxyribonucleotide at the 5’ end is a 5'-5' linked deoxythymidine and the inverted deoxyribonucleotide at the 3’ end is a 3’-3' linked deoxythymidine.
  • the sense strand comprises a nucleotide comprising the modified nucleotide component represented by Formula (I) at the 3’ end; optionally wherein R 1 is -CH3 and B 1 is uracil.
  • the modification includes one or more phosphoro thioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5’ linkages, 2'-5' linked analogs of these) having inverted polarity wherein the adjacent pairs of nucleoside
  • the modified nucleotide includes at least one of: 5 ’-vinyl phosphonate nucleotide, a 5 ’-phosphate or phosphate mimic, a locked nucleic acid (LNA), a 2’-M0E (methoxyethyl)nucleotide, and/or a 2’-arabino fluoro (2’-araF) nucleotide.
  • the modified nucleotide antisense strand comprises a phosphate mimic at the 5’ end; optionally wherein the phosphate mimic is a 5'-E-Vinyl -phosphonate or a 4'-O- phosphonate.
  • the modified nucleotide comprises at least one of: a 2’-deoxy- 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2’-amino-modified nucleotide, 2’-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and/or a non-natural base comprising nucleotide.
  • the antisense strand and/or the sense strand comprises at least one internucleoside linkage selected from the group consisting of a phosphorothioate linkage, a phosphorodithioate linkage, a phosphotriester linkage, an alkylphosphonate linkage, an aminoalkylphosphotriester linkage, an alkylene phosphonate linkage, a phosphinate linkage, a phosphoramidate linkage, a phosphoromorpholidate linkage, a phosphoropiperazidate linkage, an aminoalkylphosphoramidate linkage, a thiophosphoramidate linkage, a thionoalkylphosphonate linkage, a thionoalkylphosphotriester linkage, a thiophosphate linkage, a selenophosphate linkage, and a boranophosphate linkage.
  • a phosphorothioate linkage a phosphorodithioate linkage
  • the antisense strand and/or the sense strand comprises at least one nucleotide modified linkage. In some embodiments, all the nucleotide linkages in the antisense strand are modified linkages. In some embodiments, the antisense strand and/or the sense strand comprises at least one a phosphorothioate (PS) bond.
  • PS phosphorothioate
  • dsRNAs of the disclosure involves chemically linking to the dsRNA one or more ligand or targeting moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA (e.g., a GalNAc of the present disclosure, e.g., tri-GalNAc6)
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553- 6556), cholic acid (Manoharan et al., Biorg. Med. Chem.
  • a thioether e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306- 309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
  • Acids Res., 1990, 18:3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylaminocarbo nyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923- 937).
  • the ligand or targeting moiety e.g., a GalNAc of the present disclosure, e.g., tri-GalNAc6
  • the ligand or targeting moiety is conjugated to the 5’ end, 3’ end or both ends of the dsRNA.
  • the ligand or targeting moiety e.g., a GalNAc of the present disclosure, e.g., tri-GalNAc6
  • the 3’ end of the sense strand of the dsRNA is conjugated to the 3’ end of the sense strand of the dsRNA.
  • the ligand or targeting moiety (e.g., a GalNAc of the present disclosure, e.g., tri-GalNAc6) is conjugated to the 3’ end of the antisense strand of the modified dsRNA.
  • the ligand or targeting moiety (e.g., a GalNAc of the present disclosure, e.g., tri-GalNAc6) is conjugated to the 5’ end of the sense strand of the dsRNA.
  • the ligand or targeting moiety (e.g., a GalNAc of the present disclosure, e.g., tri-GalNAc6) is conjugated to the 5’ end of the antisense strand of the modified dsRNA.
  • the ligand or targeting moiety is at least one N- Acetyl-Galactosamine (GalNAc).
  • the dsRNA may be modified by a non-ligand group.
  • non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature.
  • Such non-ligand moieties include lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
  • 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., 1990, 18:3777 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or 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 octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Then, 1996, 277:923).
  • Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the oligonucleotide sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase.
  • the dsRNA conjugate can be purified for example by HPLC methods.
  • Conjugating a ligand to a dsRNA can enhance its cellular absorption as well as targeting to a particular tissue or uptake by specific types of cells such as liver cells.
  • a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane and or uptake across the liver cells.
  • the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis.
  • cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs.
  • Other lipophilic compounds that have been conjugated to oligonucleotides include 1 -pyrene butyric acid, l,3-bis-O-(hexadecyl)glycerol, and menthol.
  • a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate- receptor-mediated endocytosis.
  • dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor- mediated endocytosis.
  • Li and coworkers report that attachment of folic acid to the 3 ’-terminus of an oligonucleotide resulted in an 8- fold increase in cellular uptake of the oligonucleotide.
  • Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, delivery peptides and lipids such as cholesterol and cholesterylamine.
  • carbohydrate clusters include Chol-p-(GalNAc)3 (N-acetyl galactosamine cholesterol) and LCO(GalNAc)3 (N-acetyl galactosamine - 3’-Lithocholic-oleoyl).
  • a dsRNA oligonucleotide of the disclosure further comprises a carbohydrate.
  • the carbohydrate conjugated dsRNA is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein.
  • “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom.
  • Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums.
  • Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
  • a carbohydrate conjugate for use in the compositions and methods of the disclosure is a monosaccharide.
  • the monosaccharide is an N-acetylgalactosamine, of formula I or formula II such as
  • a carbohydrate is conjugated to the 5’ end, 3’ end or both ends of the modified dsRNA.
  • the ligand or targeting moiety is conjugated to the 3’ end of the sense strand of the modified dsRNA.
  • the ligand or targeting moiety is conjugated to the 3’ end of the antisense strand of the modified dsRNA.
  • the carbohydrate is at least one N-Acetyl-Galactosamine (GalNAc).
  • compositions comprising the dsRNAs targeting INHBE genes of the disclosure.
  • the disclosure provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a targeting INHBE genes, such as pathological processes mediated by targeting INHBE gene expression.
  • Such pharmaceutical compositions are formulated based on the mode of delivery.
  • compositions featured herein are administered in dosages sufficient to inhibit expression of INHBE genes.
  • treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.
  • Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the disclosure can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
  • a suitable mouse model is, for example, a mouse containing a plasmid expressing human INHBE.
  • Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human INHBE.
  • the data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of compositions featured in the disclosure lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the dsRNAs featured in the disclosure can be administered in combination with other known agents effective in treatment of pathological processes mediated by INHBE gene expression.
  • the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
  • compositions disclosed herein comprise a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions.
  • Certain delivery systems are useful for preparing pharmaceutical compositions including those comprising hydrophobic compounds.
  • certain organic solvents such as dimethylsulfoxide are used.
  • the dsRNA of the disclosure is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • dsRNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • the pharmaceutical composition comprises an excipient.
  • a “pharmaceutical carrier’' or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.
  • the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pre-gelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
  • binding agents e.g., pre-gelatinized maize starch, polyvinylpyrrolidone or
  • compositions of the present disclosure can also be used to formulate the compositions of the present disclosure.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions and formulations which include the dsRNA compositions and pharmaceutical compositions of the disclosure.
  • the dsRNA composition or pharmaceutical composition of the disclosure is administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • administration of the pharmaceutical composition is topical (including buccal and sublingual), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.
  • parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.
  • compositions containing a dsRNA of the disclosure can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration.
  • Useful formulations can be prepared by methods well known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
  • compositions for example, are sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
  • the dsRNA is delivered in a manner to target a particular tissue, for example the liver.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound (e.g., dsRNA molecule) which produces a therapeutic effect.
  • a formulation of the present disclosure comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and poly anhydrides; and a compound (e.g., dsRNA molecule) of the present disclosure.
  • an aforementioned formulation renders orally bioavailable a compound (e.g., dsRNA molecule) of the present disclosure.
  • Formulations of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound (e.g., dsRNA molecule) of the present disclosure as an active ingredient.
  • a compound (e.g., dsRNA molecule) of the present disclosure may also be administered as a bolus, electuary or paste.
  • Liquid dosage forms for oral administration of the compounds (e.g., dsRNA molecules) of the disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the disclosure provides a method for inhibiting the expression of an INHBE gene in a cell.
  • the method comprises administering a dsRNA targeting an INHBE gene to a cell, such that expression of the target INHBE gene in the cell is reduced.
  • the disclosure includes methods performed in cells in in vitro or in vivo. In some embodiments, the method is performed in the cell of an animal, e.g., a mouse, a rat, a non-human primate, or a human.
  • the present disclosure also provides methods of using a dsRNA of the disclosure and/or a composition containing an dsRNA of the present disclosure to reduce and/or inhibit INHBE expression in a cell.
  • 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 transcript of a INHBE gene, thereby inhibiting expression of the INHBE gene in the cell. Reduction in gene expression can be assessed by any methods known in the art.
  • a reduction in the expression of INHBE may be determined by determining the mRNA expression level of INHBE using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR, by determining the protein level of INHBE using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques, and/or by determining a biological activity of INHBE, such as affecting one or more molecules associated with the cellular blood clotting mechanism (or in an in vivo setting, blood clotting itself).
  • the cell may be contacted 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 a INHBE gene.
  • 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 or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell.
  • the cell is a human cell, e.g.
  • the INHBE expression is inhibited by at least 30% relative to a control after administration of the dsRNA oligonucleotide of the disclosure.
  • a method of treating a disorder mediated by INHBE comprising administering to a subject in need of such treatment a therapeutically effective amount of an dsRNA oligonucleotide or a pharmaceutical composition of the disclosure.
  • the disorder is a cardiovascular disorder. In some embodiments, the disorder is cardiovascular disease.
  • the compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization.
  • RNAi RNA interference
  • This example describes a screen for siRNA-based inhibition of the INHBE gene in a hepatocyte derived cellular carcinoma cell model (Huh-7). Briefly, Huh-7 cells were transfected were transfected with 147 3’-GalNAc conjugated, modified siRNAs (Duplexes 100488 - 100634, SEQ ID NOs: 294 - 440, sense strand; and SEQ ID NOs: 441- 587, antisense strand) at 10 nM and 0.1 nM. Sequences of exemplary, unmodified and modified siRNA compounds are shown in Table 1 and Table 2, respectively. Compounds in Table 2 were 3’ GalNac modified.
  • INHBE mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells.
  • Table 3 and FIG. 1A, B and C show the results of single dose screens at 10 nM and O. lnM in Huh7 cells using the selected INHBE siRNAs. The data are presented as percent inhibition of INHBE mRNA in the cells transfected with siRNAs relative to INHBE mRNA in the mock treated control cells.
  • This example describes a screen of exemplary INHBE siRNA compounds in primary human hepatocytes (PHH) cells in a single dose screen at lOOnM, 33nM, 1 InM, 3.7nM, 1.2nM, 0.412nM, 0.137nM, and 0.046nM of the selected siRNA (Table 2).
  • ZAHBE mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells and the average KD and SD was determined. The data are presented as percent inhibition of INHBE mRNAs in the cells treated with siRNAs relative to INHBE mRNA in the PBS control cells.
  • Table 4 and FIG. 2 show the results of the in vitro dose-response INHBE siRNA screens.
  • This example describes a screen of exemplary INHBE siRNA compounds in primary human hepatocytes (PHH) cells in a single dose screen at lOnM and InM of the selected siRNA (Table 2). INHBE mRNA level was measured by quantitative PCR and normalized to GAPDH relative to mock treated control cells and the mean KD and SD was determined.
  • mice were subcutaneously injected with INHBE siRNA compounds from Table 6 at 1 mg/kg. Three days post injection, the mice were hydrodynamically injected with a DNA plasmid encoding the full-length human INHBE transcript. One day after the injection of the plasmid, liver samples were harvested and analyzed for INHBE mRNA expression relative to mice treated with the same volume of PBS. INHBE mRNA levels were measured by quantitative PCR and normalized to NEO gene included in the plasmid used to express INHBE. The data are presented as relative gene expression of INHBE mRNA in the liver relative to PBS treated animals.
  • Tables 6-7 The modified and unmodified sense and antisense strand sequences of Compounds 100329-100341 are summarized in Tables 6-7.
  • Table 8 and FIG. 4 show the results of single dose INHBE siRNA injection in INHBE BALB/c mice. The results show, that siRNA compounds 100635, 100638, 100639, 100642, 100643, 100644, 100645 and 100646 reduce INHBE expression by more than 80%. Additionally, compounds 100635-100646 all showed improved potency relative to siRNA compound 100647.
  • Table 6 Exemplary tri-GalNAc6 or L96 conjugated, modified INHBE siRNA compounds.
  • the structures of tri-GalNAc6 and L96 ligand in the exemplary siRNA compounds are provided below:
  • siRNA nucleotide sugar wherein B is the nucleotide base uracil (nucleotide abbreviation: tmU) or cytosine (nucleotide abbreviation tmC)
  • TNA analog wherein B is a uracil base (abbreviation: utU) or an adenine base (abbreviation utA)
  • Example 5 Evaluation of knockdown of human INHBE with select siRNAs in in vivo mouse hydrodynamic injection (HDI) model
  • siRNA compounds (Compounds 100643 and 100647-100657) were tested for knockdown of human INHBE in a mouse model hydrodynamic injected with DNA plasmid encoding the full-length human INHBE transcript. Briefly, 6-7-week-old female BALB/c mice were subcutaneously injected with INHBE siRNA compounds from Table 9 at 1 or 1.5 mg/kg. Three days post injection, the mice were hydrodynamically injected with a DNA plasmid encoding the full-length human INHBE transcript. One day after the injection of the plasmid, liver samples were harvested and analyzed for INHBE mRNA expression relative to mice treated with the same volume of PBS. INHBE mRNA levels were measured by quantitative PCR and normalized to NEO gene included in the plasmid used to express INHBE. The data are presented as relative gene expression of INHBE mRNA in the liver relative to PBS treated animals.
  • FIG. 5 show the results of single dose INHBE siRNA injection in INHBE BALB/c mice. The results show, that siRNA compounds 100643, 100649, 100650, 100654, 100655, and 100657 showed improved potency relative to siRNA compound 100647.
  • Table 9 Exemplary tri-GalNAc6 or L96 conjugated, modified INHBE siRNA compounds.
  • the structures of tri-GalNAc6 and L96 ligand in the exemplary siRNA compounds are provided below:
  • Example 6 Evaluation of knockdown of INHBE with siRNAs in in vivo non-human primate model
  • FIG. 6 shows the results of single dose INHBE siRNA injection in cynomolgus monkeys. The results show that 69% knockdown (Compound 100639) and 75% knockdown (Compound 100642) was observed in animals at day 56 after injection (FIG. 6).
  • Example 7 In vitro activity and dose-response screen in hTLR7, hTLR8, and hTLR9 cells
  • This example evaluates the agonist activity of compounds in the cell-based human TLR Toll-like receptor (hTLR) reporter assay.
  • hTLR human TLR Toll-like receptor
  • siRNA compounds 100647, 100635, 100638, 100639, 100642, 100643, and 10064 were tested using commercial cellbased hTLR7, hTLR8 and hTLR9 reporter assays. Briefly, assays were performed using a 4- fold dilution from 100 nM under 9 concentrations by transfection of HEK-293 cells in duplicate, and with a duration of treatment of 24 hours.
  • R848 was purchased from a commercial vendor and used as the agonist for the hTRL7 and hTLR8 reporter assays.
  • ODN 2006 was purchased from a commercial vendor and used as the agonist for the hTRL9 reporter assays.
  • This example evaluates the RNA sequence transcriptome in primary human hepatocytes to assess potential off-target risk of exemplary siRNA compounds 100647, 100635, 100638, 100639, 100642, 100643, and 100645.
  • primary human hepatocyte (PHH) cells were collected after 48 hours of treatment with of the exemplary siRNA compounds for RNA extrations, library construction, and sequencing.
  • FIG. 8 shows the results of the RNA sequence transcriptome analysis in primary human hepatocytes treated with exemplary siRNA compounds.
  • the data are presented as a volcano plot of differentially expresses genes (DEGs) among different groups, and show that INHBE is significantly down-regulated in all groups.
  • DEGs differentially expresses genes
  • siRNA compounds 100635, 100642, and 100643 were evaluated via a non-GLP mini toxicology study in mice. Briefly, 7 week old male C57BL/6J mice (5 per group) were subcutaneously injected with a single 50 mg/kg dose of one of the 100635, 100642, and 100643 siRNA compounds (i.e. , at day 0).
  • Urine and blood samples harvested on day 0 were handled as follows. Plasma was snap frozen on snap frozen on dry ice upon collection, stored at -80 deg transferred for biochemistry analysis. Urine was stored at 4 degrees or -80 degree Celsius until transferred for biochemistry analysis.
  • Urine, blood, liver and kidney samples harvested on day 7 were handled as follows. Plasma: snap frozen on dry ice upon collection, stored at -80 deg transferred for biochemistry analysis Plasma was stored on ice until transferred for coagulation assays
  • FIG. 9 and Table 12 shows the results of injecting C57BL/6J mice with a single 50 mg/kg dose of one of 100635, 100642, or 100643 siRNA compounds.
  • FIG. 9 shows the results of the mice biochemical tests over 7 days post dosing.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TAG triglycerides
  • LDL-C low-density lipoprotein cholesterol
  • HDL-C high-density lipoprotein cholesterol
  • CREZ creatinine
  • LH lactate dehydrogenase
  • Table 12 shows the results of the liver and kidney pathology study over 7 days post dosing. The study results indicate no significant lesions (damage) in the experimental subjects. Observed changes in the PBS group may have be due to background lesions. [0217] Table 12. Liver and kidney pathology study results.
  • Example 10 Evaluation of knockdown of INHBE with Compound 100639 in a 140-day in vivo non-human primate model
  • Blood INHBE expression was evaluated by ELISA and blood biochemistry of liver enzymes were evaluated. Blood samples were collected four days before injection (D-4), 7 days after injection (D7), 14 days after injection (D14), 28 days after injection (D28), 42 days after injection (D42), 56 days after injection (D56), 70 days after injection (D70), and 84 days after injection (D84).
  • Liver biopsies were obtained 4 days prior to injection (D-4), 28 days after injection (D28), 56 days after injection (D56), 84 days after injection (D84), 112 days after injection (DI 12), and 140 days after injection (D140).
  • Liver INHBE mRNA levels were measured by quantitative PCR and normalized to the D-4 level for each individual animals. Data readouts include liver INHBE mRNA knockdown by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and liver siRNA pharmacokinetics (PK) by Stem-loop polymerase chain reaction (PCR) (antisense strand).
  • RT-qPCR reverse transcription quantitative real-time polymerase chain reaction
  • PK liver siRNA pharmacokinetics
  • PCR Stem-loop polymerase chain reaction
  • FIG. 10 shows a study scheme summarizing the protocol described above.
  • FIG. 11 shows mean gene relative expression of INHBE in subjects across days after injection of the dose of Compound 100639 at Day 0 (DO). The data were normalized to the level 4 days before injection for each individual monkey subject (D-4). The data show that INHBE knockdown is observed for 140 days: 56% at Day 28 (D28), 69% at Day 56 (D56), 62% at Day 84 (D84), 74% at Day 112 (DI 12), and 70% for Day 140 (D140).
  • Example 11 Evaluation of knockdown of INHBE with Compound 100642 in an 84-day in vivo non-human primate model
  • Blood INHBE expression was evaluated by ELISA and blood biochemistry of liver enzymes were evaluated. Blood samples were collected four days before injection (D-4), 7 days after injection (D7), 14 days after injection (D14), 28 days after injection (D28), 42 days after injection (D42), 56 days after injection (D56), 70 days after injection (D70), and 84 days after injection (D84).
  • FIG. 12 shows a study scheme summarizing the protocol described above.
  • This example evaluates the pharmacodynamics of Compound 100639 in spontaneously obese Cynomolgus monkeys. Briefly, spontaneously obese monkeys received either vehicle or Compound 100639 at 10 mg/kg (mpk) respectively on day 0 (first dose) and day 70 (second dose). Bodey weight and BMI (Body Mass Index) were measured throughout the study. Liver biopsy samples were collected to assess the expression of INHBE mRNA levels by RT-qPCR (reverse transcription quantitative real-time polymerase chain reaction) on day -14 (before dosing), day 28, day 56, day 98, and day 126. Blood samples were collected to determine INHBE protein levels by LC-MS/MS (liquid chromatography in combination with tandem mass spectrometry) on day 28.
  • RT-qPCR reverse transcription quantitative real-time polymerase chain reaction
  • FIG. 14A The data are presented as the percentage of body weight change from baseline over time (FIG. 14A), the BMI change from baseline over time (FIG. 14B), the relative level of INHBE gene expression in the liver as compared to baseline over time (FIG. 14C) and the relative percentage of INHBE protein in plasma as compared to baseline over time (FIG. 14D).
  • Example 13 In vitro dose response concentration (DRC) studies of exemplary siRNA compounds.
  • Table 13 below provides the modified siRNA compounds studied.
  • the unmodified sequences for compounds 100643, 100658, 100659, 100660, 100661, and 100662 are provided as SEQ ID NO: 593 (sense) and SEQ ID NO: 602 (antisense).
  • the unmodified sequences for compounds 100663, 100664, 100665, and 100666 are provided as SEQ ID NO: 700 (sense) and SEQ ID NO: 602 (antisense).
  • Table 14 provides the unmodified sequences of compounds 100643, 100658, 100659, 100660, 100661, 100662, 100663, 100664, 100665, and 100666.
  • FIG. 15 summarizes the concentrations and conditions used. Table 15 provides ICso and maximal inhibition values for the exemplary compounds.
  • FIGS. 16A-16J show percent inhibition INHBE dose-response plots of siRNA compounds of Table 13. [0233] Table 13. Exemplary tri-GalNAc6 modified INHBE siRNA compounds. The structure of tri-GalNAc6 in the exemplary siRNA compounds is provided below:
  • TNA analog wherein B is the nucleotide base adenine (TNA analog abbreviation utA)
  • Example 14 Evaluation of knockdown of human INHBE with select siRNAs in in vivo human INHBE transgenic mouse model
  • mice received either vehicle (PBS) or INHBE siRNA compound at 3 mg/kg (mpk) on day 0. Animals were sacrificed and liver samples were collected on day 14. INHBE knockdown effect was determined by RT-qPCR (reverse transcription quantitative real-time polymerase chain reaction).
  • FIG. 17 shows the results of a single 3 mpk dose of INHBE siRNA injection in human INHBE transgenic mice. The results show that INHBE siRNA compounds 100643, 100660, 100661, and 100662 reduced human INHBE gene expression in liver relative to mice treated with vehicle.
  • Example 15 Preparation of GalNAc-6-amidite and GalNAc-6-CPG
  • Step 2 To a solution of 2 (54.0 g, 440.0 mol) in pyridine (1.1 L) was added at r.t. in N2 atmosphere. Next DMT-C1 (140.2 g, 410.0 mmol) was added at 0 °C in the mixture. Then the reaction mixture was stirred at r.t. for 5 hours. The LCMS showed 2 was consumed.
  • Step 3 To a solution of 3 (170.0 g, 400.0 mmol) in 1.4-dioxane (1.7 L), 5.0 M sodium hydroxide solution was added dropwise. The solution was stirred overnight at 40 °C and was then diluted with ethyl acetate. The product was extracted with water (500.0 *4mL). The organic layer was washed with brine and dried over Na2SO4. Then the solution was concentrated under reduced pressure to give 4 (176.0 g, 90% purity) as solid which was used directly for the next step.
  • ESI-LCMS m/z 389.4 [M-H]'.
  • ESI- LCMS m/z 406.2 [M+H] + .
  • Step 5 Compound 5a (24.0 g, 46.6 mmol) was added in DMF (190.0 mL). Next EDCI (11.6 g, 61.0 mmol) and HOBT (8.2 g, 61.0 mmol) was added in the mixture at r.t. for the 20 min. Then the DIPEA (12 g, 93.0 mmol) and compound 5 (19.0 g, 46.6 mmol) was added in the mixture at r.t. Then the reaction mixture was stirred at r.t. for 17 h. The LCMS showed compound 5 was consumed. The reaction mixture extracted with DCM (600.0 mL *5). The organic layer was washed with brine and dried over Na2SC>4.
  • Step 6 (GalNAc-6-amidite): To a solution of 6 (14.0 g, 15.6 mmol) in dichloromethane (140.0 mL) with an inert of nitrogen were added CEOP[N(iPr)2]2 (5.6 mL, 18.8 mmol) and DCI (1.66 g, 14.1 mmol) in order at room temperature. The resulting solution was stirred for 1 h at room temperature and diluted with 50.0 mL dichloromethane and washed with 50.0 mL *2 of saturated aqueous sodium bicarbonate and 50.0 mL of saturated aqueous sodium chloride respectively.
  • the suspension of resin was added to the mixture of pyridine and Ac 2 O (5/1, 6 V in total). The suspension was agitated by mechanism stir at 40 °C for 4 h. The reaction solution was filtered and the filter cake rinsed successively with DCM (8 V * 4). The filter cake was dried at 30 °C under reduced pressure to get the solid to a fine powder.
  • siRNA conjugates of tri-GalNAc6 were prepared from GalNAc6-CPG, with the following procedure:
  • Sense and antisense strand sequences of siRNAs were synthesized using oligonucleotide synthesizers following a standard solid phase synthesis protocol based on phosphoramidite chemistry.
  • the ligand-CPG succinates were used as solid supports continued by two incorporations of the ligand phosphoramidites.
  • 3 ’-conjugation three consecutive incorporations of the corresponding GalNAc phosphoramidites were performed.
  • solid support bound oligomer was cleaved together by treatment with ammonia for 12 hours at 55 °C (24 h in the case of tri-GalNAc5).
  • IP- RPHPLC ion pairing reversed phase HPLC
  • Purified single strand oligonucleotide product from IP-RP-HPLC was converted to sodium salt by addition of NaOAc followed by desalting. Annealing of equimolar complementary sense stand and antisense strand oligonucleotide was carried out in nuclease- free water to form the double strand siRNAs, followed by a lyophilization procedure.

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Abstract

La divulgation concerne un acide ribonucléique double brin (ARNdb) ciblant un gène INHBE, ainsi que des méthodes d'utilisation de l'ARNdb permettant d'inhiber l'expression d'INHBE .
PCT/US2025/019447 2024-03-11 2025-03-11 Compositions et méthodes d'inhibition de l'expression de gènes de la sous-unité bêta de l'inhibine (inhbe) Pending WO2025193754A2 (fr)

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PCT/US2024/019416 WO2024187190A2 (fr) 2023-03-09 2024-03-11 Compositions et procédés pour l'inhibition de l'expression de gènes de la sous-unité bêta de l'inhibine (inhbe)
USPCT/US2024/019416 2024-03-11
US202463677596P 2024-07-31 2024-07-31
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