Compositions and methods for inhibiting the expression of the inhibin subunit beta E (INHBE) gene

Abstract

This disclosure relates to a double-stranded ribonucleic acid (dsRNA) that targets the INHBE gene, and a method for inhibiting INHBE expression using the dsRNA.

Description

[Technical Field]

[0001] cross reference This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 489,325, filed on 9 March 2023, the disclosure of which is incorporated herein by reference in its entirety. Sequence List

[0002] This application includes an electronically submitted sequence listing XML, which is incorporated herein by reference in its entirety. A copy of the XML was created on XX / XX / 20XX, named XXXXX, and has a size of XXX,XXX bytes.

[0003] This disclosure relates to a double-stranded ribonucleic acid (dsRNA) that targets the INHBE gene, and a method for inhibiting intracellular INHBE expression using the dsRNA. [Overview of the project]

[0004] This disclosure is based in part on the development of double-stranded ribonucleic acid (dsRNA) targeting the inhibin subunit beta E (INHBE) gene, a pharmaceutical composition comprising the dsRNA targeting the INHBE gene, and a method for inhibiting intracellular INHBE expression using the dsRNA.

[0005] In certain embodiments, the Disclosure relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting INHBE expression, comprising a sense strand and an antisense strand, each having a length of 15 to 30 nucleotides, 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, and (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 (c) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 590, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 600, and (d) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 601, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 592, and (e) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 6 (f) The sense strand contains a sequence that is at least 70% or 80% identical to the sequence of sequence 02, and the sense strand contains a sequence that is at least 70% or 80% identical to the sequence of sequence number 593, (g) The antisense strand contains a sequence that is at least 70% or 80% identical to the sequence of sequence number 603, and the sense strand contains a sequence that is at least 70% or 80% identical to the sequence of sequence number 594, (g) The antisense strand contains a sequence that is at least 70% or 80% identical to the sequence of sequence number 604, and the sense strand contains a sequence that is at least 70% or 80% identical to the sequence of sequence number 595 The present invention provides a double-stranded ribonucleic acid (dsRNA) comprising, (h) an antisense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 605, and a sense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 596, or (i) an antisense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 606, and a sense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 597.In certain embodiments, the Disclosure relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting INHBE expression, wherein the dsRNA comprises a sense strand and an antisense strand, each being 15 to 30 nucleotides in length, the antisense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 607, (b) the antisense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 617, and the sense strand comprising (c) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 608, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 618, and (d) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 619, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 610, and (e) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of sequence number 620 (f) The sense strand includes a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 611, and (g) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 621, and the sense strand includes at least 15 consecutive nucleotides of the sense strand sequence including the sequence of SEQ ID NO: 612, and (g) The antisense strand includes at least 15 consecutive nucleotides of the antisense strand sequence including the sequence of SEQ ID NO: 622, and the sense strand includes the sequence of SEQ ID NO: 613 The present invention provides a double-stranded ribonucleic acid (dsRNA) comprising a sequence that is at least 70% or 80% identical to the sequence of (h) the antisense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 623, and the sense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 614, or (i) the antisense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 624, and the sense strand comprising a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 615.

[0006] In some embodiments, the Disclosure relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting INHBE expression, comprising a sense strand and an antisense strand, each having a length of 15 to 30 nucleotides, wherein (a) the antisense strand comprises at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 598, and the sense strand comprises at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 589, and (b) the antisense strand comprises at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 599. (c) The sense strand includes at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 590, (d) The antisense strand includes at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 600, and the sense strand includes at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 591, (e) The antisense strand comprises at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 602, and the sense strand comprises at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 593, (f) The antisense strand comprises at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 603, and the sense strand comprises at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 594, and (g) The antisense strand comprises an antisense strand containing the sequence of SEQ ID NO: 604 (h) The sense strand comprises at least 15 consecutive nucleotides of the sense strand sequence, and the sense strand comprises at least 15 consecutive nucleotides of the sense strand sequence comprising the sequence of SEQ ID NO: 595, or (i) The antisense strand comprises at least 15 consecutive nucleotides of the antisense strand sequence comprising the sequence of SEQ ID NO: 605, and the sense strand comprises at least 15 consecutive nucleotides of the sense strand sequence comprising the sequence of SEQ ID NO: 596, orThe present invention provides a double-stranded ribonucleic acid (dsRNA) comprising at least 15 consecutive nucleotides in a sense strand sequence containing the sequence of sequence number 597. In certain embodiments, the Disclosure relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting INHBE expression, 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 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 616, and the sense strand comprises at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 607; (b) the antisense strand comprises at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 617, and the sense strand comprises at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 608; (c) the antisense strand comprises at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 618, and the sense strand comprises at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 609; (d) the antisense strand comprises at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 619, and the sense strand comprises the sequence (e) The antisense strand includes at least 15 consecutive nucleotides of a sense strand sequence containing sequence number 610, and the sense strand includes at least 15 consecutive nucleotides of an antisense strand sequence containing sequence number 620, and the sense strand includes at least 15 consecutive nucleotides of a sense strand sequence containing sequence number 611, (f) The antisense strand includes at least 15 consecutive nucleotides of an antisense strand sequence containing sequence number 621, and the sense strand includes at least 15 consecutive nucleotides of a sense strand sequence containing sequence number 612, (g) The antisense strand includes at least 15 consecutive nucleotides of an antisense strand sequence containing sequence number 622, and the sense strand includes at least 15 consecutive nucleotides of a sense strand sequence containing sequence number 613, or (h) The antisense strand includes at least 15 consecutive nucleotides of an antisense strand sequence containing sequence number 623, and the sense strand includes at least 15 consecutive nucleotides of a sense strand sequence containing sequence number 614, or (i) The antisense strand isThe present invention provides a double-stranded ribonucleic acid (dsRNA) comprising at least 15 consecutive nucleotides of an antisense strand sequence containing the sequence of SEQ ID NO: 624, and a sense strand comprising at least 15 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 615.

[0007] In some embodiments, the INHBE gene is human INHBE. In some embodiments, INHBE is human INHBE containing the sequence shown in SEQ ID NO: 588 (NM_031479.5).

[0008] In some embodiments, the sense strand is 70%, 80%, 90%, 95%, or more identical to the sense strand sequence containing 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: 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, or SEQ ID NO: 615. In some embodiments, the sense strand includes at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of a sense strand sequence that includes 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: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ 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. In some embodiments, the sense strand comprises (a) 20 consecutive nucleotides of a sense strand sequence including 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: 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, or SEQ ID NO: 615, and (b) SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, (c) comprising 21 consecutive nucleotides of a sense strand sequence containing the sequence of sequence number 597, sequence number 612, sequence number 613, sequence number 614, or sequence number 615; (c) comprising 22 consecutive nucleotides of a sense strand sequence containing the sequence of sequence number 594, sequence number 595, sequence number 596, sequence number 597, sequence number 612, sequence number 613, sequence number 614, or sequence number 615; and / or (d) comprising 23 consecutive nucleotides of a sense strand sequence containing the sequence of sequence number 594 or sequence number 612.In some embodiments, the antisense strand includes at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of an antisense sense strand sequence that includes 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, or SEQ ID NO: 624. In some embodiments, the antisense strand comprises (a) 21 consecutive nucleotides of an antisense sense strand sequence including 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, or SEQ ID NO: 624, and (b) 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, sequence (c) comprising 22 consecutive nucleotides of an antisense sense strand sequence containing the sequence of sequence number 617, sequence number 618, sequence number 619, sequence number 620, sequence number 621, sequence number 622, sequence number 623, or sequence number 624, and / or (c) comprising 23 consecutive nucleotides of an antisense sense strand sequence containing the sequence of sequence number 598, sequence number 599, sequence number 600, sequence number 601, sequence number 602, sequence number 603, sequence number 604, sequence number 605, sequence number 606, sequence number 616, sequence number 617, sequence number 618, sequence number 619, sequence number 620, sequence number 621, sequence number 622, sequence number 623, or sequence number 624.In some embodiments, the sense strand sequence is selected from a sense strand sequence including 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: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ 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, and the antisense strand is selected from an antisense strand sequence including 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, or SEQ ID NO: 624. In some embodiments, the sense strand sequence is selected from a sense strand sequence including 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: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ 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. In some embodiments, the antisense strand is selected from an antisense strand sequence including 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, or SEQ ID NO: 624. In some embodiments, the antisense strand includes the sequence of sequence number 598, and the sense strand includes the sequence of sequence number 589. In some embodiments, the antisense strand includes the sequence of sequence number 599, and the sense strand includes the sequence of sequence number 590. In some embodiments, the antisense strand includes the sequence of sequence number 600, and the sense strand includes the sequence of sequence number 591. In some embodiments, the antisense strand includes the sequence of sequence number 601, and the sense strand includes the sequence of sequence number 592.In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 602, and the sense chain includes the sequence of SEQ ID NO: 593. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 603, and the sense chain includes the sequence of SEQ ID NO: 594. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 604, and the sense chain includes the sequence of SEQ ID NO: 595. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 605, and the sense chain includes the sequence of SEQ ID NO: 596. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 606, and the sense chain includes the sequence of SEQ ID NO: 597. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 616, and the sense chain includes the sequence of SEQ ID NO: 607. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 617, and the sense chain includes the sequence of SEQ ID NO: 608. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 618, and the sense chain includes the sequence of SEQ ID NO: 609. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 619, and the sense chain includes the sequence of SEQ ID NO: 610. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 620, and the sense chain includes the sequence of SEQ ID NO: 611. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 621, and the sense chain includes the sequence of SEQ ID NO: 612. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 622, and the sense chain includes the sequence of SEQ ID NO: 613. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 623, and the sense chain includes the sequence of SEQ ID NO: 614. In some embodiments, the antisense chain includes the sequence of SEQ ID NO: 624, and the sense chain includes the sequence of SEQ ID NO: 615. In some embodiments, at least one nucleotide of the dsRNA is a 5'-vinylphosphonate nucleotide, a 2'-O-methyl modified nucleotide, an inverted deoxyribonucleotide (3'-3' linked nucleotide or 5'-5' linked nucleotide), a nucleotide containing a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide, or a nucleotide containing a modified nucleotide component represented by the following formula (I): [ka] and a nucleotide comprising a modified nucleotide component represented by the following formula (II): [Chemical formula] wherein B 1 and B 2 each is a nucleobase, and R 1 is selected from the group consisting of hydrogen and C 1-6 alkyl, and is a modified nucleotide selected from the group consisting of nucleotides, and optionally the antisense strand and the sense strand each contain at least one modified nucleotide.

[0009] In some embodiments, the antisense strand has a 3'-terminal nucleotide overhang compared to the sense strand. In some embodiments, the 3'-terminal nucleotide overhang contains 1, 2, or 3 nucleotides compared to the sense strand. In some embodiments, the antisense strand 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 contain at least one, at least two, at least three, or at least four mismatched nucleotides. In some embodiments, the antisense strand contains a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the target mRNA corresponding to the fragment of INHBE mRNA. In some embodiments, the antisense strand of the dsRNA contains at least 80% complementarity with the fragment of INHBE mRNA. In some embodiments, the antisense strand of the dsRNA contains one, two, three, or four mismatches with respect to the fragment of INHBE mRNA. In some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide, an inverted base dropout nucleotide, a thymidine-glycol nucleic acid (GNA) S-isomer, inosine, and an inverted deoxyribonucleotide (3'-3' linked nucleotide or 5'-5' linked nucleotide), a thymidine-glycol nucleic acid (GNA) S-isomer, a nucleotide containing a modified nucleotide component represented by the following formula (I): [Chemical formula] and a nucleotide containing a modified nucleotide component represented by the following formula (II): [Chemical formula] wherein B 1 and B 2Each of which is a nucleobase, R 1 is selected from the group consisting of hydrogen and C 1-6 At least one modified nucleotide selected from the group consisting of nucleotides, which is selected from the group consisting of hydrogen and C 1 alkyl, and optionally the antisense strand and the sense strand each contain at least one modified nucleotide and a nucleotide containing a modified nucleotide component represented by formula (II). In some embodiments, B 2 and B 1 are each independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof. In some embodiments, B 2 and B 1 are each independently selected from adenine, uracil, cytosine, and modified analogs thereof. In some embodiments, R 1-6 is C 1 alkyl. In some embodiments, R 1 is -CH3. In some embodiments, B 1 is uracil. In some embodiments, R 1 is -CH3 and B 2 is uracil. In some embodiments, B 2 is adenine. In some embodiments, B 1 is uracil. In some embodiments, the sense strand contains an inverted deoxyribonucleotide at the 5' end, and optionally the inverted deoxyribonucleotide is 5'-5' linked deoxythymidine. In some embodiments, the sense strand contains an inverted deoxyribonucleotide at the 3' end, and optionally the inverted deoxyribonucleotide is 3'-3' linked deoxythymidine. In some embodiments, the sense strand contains an inverted deoxyribonucleotide at the 5' end and an inverted deoxyribonucleotide at the 3' end, and optionally the inverted deoxyribonucleotide at the 5' end is 5'-5' linked deoxythymidine and the inverted deoxyribonucleotide at the 3' end is 3'-3' linked deoxythymidine. In some embodiments, the sense strand contains a nucleotide containing a modified nucleotide component represented by formula (I) at the 3' end, and optionally in the formula R1 That is Uracil.

[0010] In some embodiments, the modified nucleotide is at least one of 5'-vinylphosphonate nucleotide, 5'-phosphate or phosphate mimetic, locked nucleic acid (LNA), 2'-MOE (methoxyethyl) nucleotide, and / or 2'-arabinofluoro (2'-araF) nucleotide. In some embodiments, the antisense chain contains a phosphate mimetic at the 5' end, optionally the phosphate mimetic being 5'-E-vinyl-phosphonate or 4'-O-phosphonate. In some embodiments, the modified nucleotide is at least one of nucleotides containing 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, base-dropped nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, and / or non-natural base.

[0011] In some embodiments, the antisense and / or sense strand includes at least one internucleoside bond selected from the group consisting of phosphorothioate bonds, phosphorodithioate bonds, phosphotryester bonds, alkylphosphonate bonds, aminoalkylphosphotryester bonds, alkylenephosphonate bonds, phosphine bonds, phosphoramidate bonds, phosphormololate bonds, phosphoropiperazidate bonds, aminoalkylphosphoamidate bonds, thiophosphoamidate bonds, thionoalkylphosphonate bonds, thionoalkylphosphotryester bonds, thiophosphate bonds, selenophosphate bonds, and boranophosphate bonds. In some embodiments, the antisense and / or sense strand includes at least one nucleotide modification bond. In some embodiments, all nucleotide bonds in the antisense strand are modification bonds. In some embodiments, the antisense and / or sense strand includes at least one phosphorothioate (PS) bond.

[0012] In some embodiments, 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. In some embodiments, the ligand or targeting moiety is conjugated to the 5' end of the sense strand of the dsRNA. In some embodiments, the ligand or targeting moiety is at least one N-acetyl-galactosamine (GalNAc). In some embodiments, the ligand or targeting moiety is expressed by the following formula (I): [ka] or represented by a pharmaceutically acceptable salt thereof, in the formula A 1 This is the binding site to dsRNA, and T 1 and T 2 Each instance, is independently selected from 5-membered heterocyclines and alkylenes, X is selected each instance from the group consisting of -OH and -SH, and L is a linker each instance. A is either nonexistent or a linker, and n is an integer from 1 to 6. In some embodiments, T 1 and T 2 Each instance independently contains a 5-membered heterocycline and C with at least one ring oxygen. 1-6 Selected from alkylenes. In some embodiments, the compound is of the following formula (IA): [ka] or represented by a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by the following formula (IAI): [ka] or represented by a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by the following formula (IA-II): [ka] or a pharmaceutically acceptable salt thereof, where a and b are integers from 1 to 20, each occurrence. In some embodiments, the ligand or targeting moiety is tri-GalNAc6. In some embodiments, the ligand or targeting moiety is L96.

[0013] In some embodiments, cells containing the dsRNA of the Disclosure are provided. In some embodiments, a vector encoding at least one unmodified strand of the dsRNA of the Disclosure, or optionally both strands, is provided. The Disclosure provides cells containing the vector.

[0014] In some embodiments, a pharmaceutical composition for inhibiting INHBE expression is provided, comprising the dsRNA of the present disclosure and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

[0015] In some embodiments, a method is provided for inhibiting intracellular INHBE expression, the method comprising (a) contacting cells with the dsRNA of the Disclosure or the pharmaceutical composition of the Disclosure, and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the INHBE gene, thereby inhibiting intracellular INHBE gene expression, the method optionally being in vivo. In some embodiments, INHBE expression is inhibited by at least 30% compared to a control.

[0016] In some embodiments, a method is provided for treating an INHBE-mediated or INHBE-associated disorder, the method comprising administering a therapeutically effective amount of the dsRNA of the Disclosure or the pharmaceutical composition of the Disclosure to a subject in need of such treatment. In some embodiments, the disorder is a cardiovascular disorder. In some embodiments, the disorder is a cardiovascular disease. [Brief explanation of the drawing]

[0017] Novel features of the present invention are described in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description which includes exemplary embodiments and accompanying drawings in which the principles of the present invention are utilized.

[0018] [Figure 1A] Figures 1A and 1B show bar graphs of the percentage inhibition rate (%) of INHBE mRNA in Huh-7 cells transfected with the GalNAc-conjugated modified siRNA shown, at 10 nM and 0.1 nM, compared to INHBE mRNA in mock-treated cells. INHBE mRNA levels were measured by quantitative PCR and normalized to GAPDH. [Figure 1B] Figures 1A and 1B show bar graphs of the percentage inhibition rate (%) of INHBE mRNA in Huh-7 cells transfected with the GalNAc-conjugated modified siRNA shown, at 10 nM and 0.1 nM, compared to INHBE mRNA in mock-treated cells. INHBE mRNA levels were measured by quantitative PCR and normalized to GAPDH. [Figure 2-1] Figure 2 shows graphs of exemplary INHBE siRNA compounds in the Huh7 cell line during single-dose screening of selected siRNAs at 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM. INHBE mRNA levels were measured by quantitative PCR, normalized to GAPDH compared to mock-treated control cells, and mean KD and SD were determined. [Figure 2-2] Figure 2 shows graphs of exemplary INHBE siRNA compounds in the Huh7 cell line during single-dose screening of selected siRNAs at 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM. INHBE mRNA levels were measured by quantitative PCR, normalized to GAPDH compared to mock-treated control cells, and mean KD and SD were determined. [Figure 2-3] Figure 2 shows graphs of exemplary INHBE siRNA compounds in the Huh7 cell line during single-dose screening of selected siRNAs at 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM. INHBE mRNA levels were measured by quantitative PCR, normalized to GAPDH compared to mock-treated control cells, and mean KD and SD were determined. [Figure 3] Figure 3 shows bar graphs of the percentage knockdown rates (%) of INHBE mRNA in human hepatocytes treated with the GalNAc-conjugated modified siRNA shown, at 10 nM and 1 nM, compared to INHBE mRNA in PBS-treated cells. INHBE mRNA levels were measured by quantitative PCR and normalized to GAPDH. [Figure 4] Figure 4 shows a graph of the relative expression of human INHBE mRNA in a hydrodynamic injection model using the 13 conjugated modified siRNAs shown, at 1 mg / kg, compared to INHBE expression in PBS-treated mice. INHBE mRNA levels were measured by quantitative PCR and normalized for NEO. [Figure 5] Figure 5 shows graphs of the relative expression of human INHBE mRNA in hydrodynamic injection models using the 12 conjugated modified siRNAs shown, at 1 mg / kg or 1.5 mg / kg, compared to INHBE expression in PBS-treated mice. INHBE mRNA levels were measured by quantitative PCR and normalized for NEO. [Figure 6] Figure 6 shows bar graphs of relative INHBE mRNA expression in non-human primate models treated with siRNA compounds A and B at 5 mg / kg. INHBE mRNA levels were measured via quantitative PCR using liver biopsy samples and normalized to INHBE expression levels at day 4 for each individual animal. [Figure 7A]Figures 7A–7C show graphs illustrating the agonist activity of the test compounds in cell-based hTLR7 reporter assays (Figure 7A), hTLR8 reporter assays (Figure 7B), and hTLR9 reporter assays (Figure 7C). For each graph, the y-axis represents the activity level as a magnification change relative to unstimulated cells. The x-axis represents the concentration of each compound in nM (log10 scale). [Figure 7B] Figures 7A–7C show graphs illustrating the agonist activity of the test compounds in cell-based hTLR7 reporter assays (Figure 7A), hTLR8 reporter assays (Figure 7B), and hTLR9 reporter assays (Figure 7C). For each graph, the y-axis represents the activity level as a magnification change relative to unstimulated cells. The x-axis represents the concentration of each compound in nM (log10 scale). [Figure 7C] Figures 7A–7C show graphs illustrating the agonist activity of the test compounds in cell-based hTLR7 reporter assays (Figure 7A), hTLR8 reporter assays (Figure 7B), and hTLR9 reporter assays (Figure 7C). For each graph, the y-axis represents the activity level as a magnification change relative to unstimulated cells. The x-axis represents the concentration of each compound in nM (log10 scale). [Figure 8-1] Figure 8 shows volcano plots of differentially expressed genes (DEGs) between different groups of primary human hepatocytes (PHH) treated with the illustrative siRNA compounds shown. The x-axis represents log² (magnitude change), and the y-axis represents the statistical significance of each gene. [Figure 8-2] Figure 8 shows volcano plots of differentially expressed genes (DEGs) between different groups of primary human hepatocytes (PHH) treated with the illustrative siRNA compounds shown. The x-axis represents log² (magnitude change), and the y-axis represents the statistical significance of each gene. [Figure 9-1]Figure 9 shows graphs of biochemical tests in mice over 7 days after receiving a single dose of PBS control or siRNA compound 100635, 100642, or 100643. Mean plasma concentrations 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), urinary total microprotein (UP), and plasma urea (UREA) are shown. [Figure 9-2] Figure 9 shows graphs of biochemical tests in mice over 7 days after receiving a single dose of PBS control or siRNA compound 100635, 100642, or 100643. Mean plasma concentrations 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), urinary total microprotein (UP), and plasma urea (UREA) are shown. [Modes for carrying out the invention]

[0019] Details of one or more embodiments of the present invention are described below. Other features, purposes, and advantages of the present invention will become apparent from the specification, drawings, and claims.

[0020] This disclosure provides dsRNA oligonucleotides for inhibiting the expression of lipoprotein (A) (INHBE) genes in cells or mammals, and methods for using such dsRNA oligonucleotides, which target the INHBE gene. This disclosure also provides compositions and methods for treating pathological conditions and diseases in mammals caused by INHBE gene expression, such as cardiovascular disease. INHBE dsRNA oligonucleotides are responsible for sequence-specific degradation of INHBE mRNA.

[0021] I. Definition For convenience, the meanings of certain terms and phrases used in this specification, the examples, and the appended claims are provided below. In the event of any obvious inconsistency between the use of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[0022] As used herein, all numbers or numerical ranges include all integers within and encompassing such ranges, or fractions of values ​​or integers within and encompassing such ranges, unless the context otherwise clearly indicates. Thus, for example, a reference to the range 90-100% includes not only 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., but also 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., as well as 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another embodiment, the reference to the range of 1 to 5,000 times includes not only 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, or 20x, but also 1.1x, 1.2x, 1.3x, 1.4x, or 1.5x, 2.1x, 2.2x, 2.3x, 2.4x, or 2.5x, etc.

[0023] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the articles. For example, "an element" means one element, or more than one element, such as multiple elements.

[0024] The term "including" is used herein to mean "including, but not limited to," and is interchangeable with it.

[0025] The term “approximately” is used herein to mean a typical acceptable range in the art. For example, “approximately” may be understood as approximately two standard deviations from the mean. In a particular embodiment, approximately is the mean ± 10%. In a particular embodiment, approximately is the mean ± 5%. When “approximately” precedes a series of numbers or ranges, it is understood that “approximately” may modify each of the series of numbers or ranges.

[0026] The term "at least" preceding a number or range of numbers is understood, as is clear from the context, to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides out of a 21-nucleotide nucleic acid molecule" means that 19, 20, or 21 nucleotides have the stated characteristic. When "at least" is present before a range of numbers or ranges, it is understood that "at least" may modify each of the numbers or ranges.

[0027] As used herein, “less than” or “less than” is understood, where logically, from the context, to zero, the value adjacent to the phrase and any value or integer less than or equal to it. For example, a double strand with an overhang of “less than or equal to 2 nucleotides” has an overhang of 2, 1, or 0 nucleotides. If “less than” precedes a series of numbers or a range, it is understood that “less than” can modify each of the numbers in the series or range. As used herein, a range includes both its upper and lower limits.

[0028] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" are used interchangeably herein and refer to deoxyribonucleotides, where the nucleic acid base is thymine, for example, deoxyribothymine. However, it will be understood that the terms "ribonucleotide," "nucleotide," or "deoxyribonucleotide" may also refer to modified nucleotides or alternative substitutions, as will be further detailed below. Those skilled in the art will be well aware that guanine, cytosine, adenine, and uracil can be substituted by other parts without substantially altering the base-pairing properties of oligonucleotides containing such substitutions. For example, but not limited to, nucleotides containing inosine as their base can base-pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be substituted in the nucleotide sequences of this disclosure with, for example, nucleotides containing inosine. An array containing such a substitution is an embodiment of the present disclosure.

[0029] "INHBE" refers to the inhibin subunit beta E gene. According to the NCBI NLM website, this gene encodes a member of the TGF-beta (transforming growth factor-beta) superfamily of proteins. The encoded preproprotein is proteolytically processed to produce the inhibin beta subunit. Inhibin is involved in regulating numerous cellular processes, including cell proliferation, apoptosis, immune responses, and hormone secretion. This gene can be upregulated under endoplasmic reticulum stress, and this protein can inhibit cell proliferation and growth in the pancreas and liver. The human INHBE mRNA sequence is GenBank acceptance number NM_031479.5 and is included herein as SEQ ID NO 588. The rhesus monkey (Macaca mulatta) INHBE mRNA sequence is GenBank acceptance number XM_028847001.1.

[0030] As used herein, “target sequence” refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the INHBE gene, including mRNA, which is the product of RNA processing of the primary transcript.

[0031] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a chain of nucleotides described by a sequence referred to using standard nucleotide nomenclature.

[0032] As used herein, and unless otherwise indicated, the term “complementary” means the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double-stranded structure, as will be understood by those skilled in the art when used to describe a first nucleotide sequence in relation to a second nucleotide sequence.

[0033] For example, a first nucleotide sequence may be described as complementary to a second nucleotide sequence when the two sequences hybridize (e.g., anneal) under stringent hybridization conditions. Hybridization conditions include the temperature, ionic strength, pH, and organic solvent concentration of the annealing and / or washing steps. The term stringent hybridization conditions refers to conditions under which a first nucleotide sequence preferentially hybridizes to its target sequence, e.g., a second nucleotide sequence, and less to other sequences, or does not hybridize at all. Stringent hybridization conditions are sequence-dependent and vary under different environmental parameters. Generally, stringent hybridization conditions involve the thermal melting point (T) of the nucleotide sequence at a given ionic strength and pH. m It is selected to be approximately 5°C lower than ). m This is the temperature (under specified ionic strength and pH) at which 50% of the first nucleotide sequence hybridizes to a perfectly matched target sequence. Extensive guidelines for nucleic acid hybridization can be found, for example, in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology -- Hybridization with Nucleic Acid Probes part I, chap. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, NY ("Tijssen").

[0034] Other conditions, such as physiologically relevant conditions that may be encountered within living organisms, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences, according to the final application of the hybridized nucleotides.

[0035] This includes base pairing of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence spanning the full length of both the first and second nucleotide sequences. Such sequences may be referred to herein as “fully complementary.” However, where the first sequence is referred to herein as “substantially complementary” with respect to the second sequence, the two sequences may be fully complementary, or they may form one or more, but generally four, three, or two or fewer, mismatched base pairs during hybridization, while retaining the ability to hybridize under conditions most relevant to their final application. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs are not considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of length 21 nucleotides and another oligonucleotide of length 23 nucleotides, where the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, may still be referred to as “fully complementary” for the purposes described herein.

[0036] As used herein, “complementary” sequences may also include, or be formed entirely from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, insofar as they satisfy the above requirements regarding their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairs or Hoogsteen-type base pairs.

[0037] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein may be used in relation to base matching between the sense strand and antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood from the context of their use.

[0038] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding INHBE), including the 5'UTR, open reading frame (ORF), or 3'UTR. For example, a polynucleotide is complementary to at least a portion of INHBE mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding INHBE.

[0039] In one embodiment, the antisense strand of the dsRNA is sufficiently complementary to the target mRNA to cause cleavage of the target mRNA.

[0040] As used herein, the term “double-stranded RNA” or “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, as defined above. Generally, the majority of the nucleotides in each strand are ribonucleotides, but each or both strands may also contain at least one non-ribonucleotide, e.g., deoxyribonucleotide and / or modified nucleotide, as described in detail herein. Furthermore, as used herein, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and all types of modifications disclosed herein or known in the art. As used in siRNA-type molecules, any such modifications are encompassed by “dsRNA” for the purposes of this specification and the claims.

[0041] The two strands forming a double-stranded structure may be different parts of a larger RNA molecule, or they may be separate RNA molecules. When the two strands are parts of a larger molecule and are therefore joined by an unbroken chain of nucleotides between the 3' end of one strand and the 5' end of the other strand forming the double-stranded structure, the connecting RNA strands are called a “hairpin loop.” When the two strands are covalently joined by means other than an unbroken chain of nucleotides between the 3' end of one strand and the 5' end of the other strand forming the double-stranded structure, the connecting structure is called a “linker.” RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, dsRNA may contain one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to the dsRNA described above.

[0042] As used herein, “nucleotide overhang” refers to an unpaired nucleotide that protrudes from the double-stranded structure of a dsRNA, such that the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. “Blunt” or “smooth-ended” means that there is no unpaired nucleotide at the end of the dsRNA, i.e., there is no nucleotide overhang. A “smooth-ended” dsRNA is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule.

[0043] The term “antisense strand” refers to a strand of dsRNA containing a region substantially complementary to the target sequence. As used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to the sequence as defined herein, e.g., the target sequence. If the complementary region is not perfectly complementary to the target sequence, the mismatch is most tolerable in the terminal region, and if present, is generally within 6, 5, 4, 3, or 2 nucleotides of the terminal region, e.g., the 5' and / or 3' ends.

[0044] As used herein, the term “sense strand” refers to a strand of dsRNA that contains a region substantially complementary to the antisense strand region.

[0045] Where referring to dsRNA, “introducing into cells” means facilitating uptake or absorption into cells, as understood by those skilled in the art. Absorption or uptake of dsRNA may occur via unassisted diffusion or activated cellular processes, or by auxiliary substances or devices. The meaning of this term is not limited to cells in vitro, and dsRNA can also be “introduced into cells,” where cells are part of a living organism. In such examples, introduction into cells would include delivery to an organism. For example, for in vivo delivery, dsRNA may be injected into a tissue site or administered systemically. Introduction into cells in vitro includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein or are publicly known in the art.

[0046] The terms “silence,” “inhibit the expression of,” “downregulate the expression of,” and “suppress the expression of,” insofar as they refer to the INHBE gene, mean in this specification at least partial suppression of INHBE gene expression, which is manifested by a reduction in the amount of mRNA that can be isolated from a first cell or cell population that has been transcribed and treated to inhibit INHBE gene expression, and this reduction is compared to a second cell or cell population (control cell) that is substantially identical to the first cell or cell population but has not been treated in the same way. The degree of inhibition is usually expressed as follows:

[0047]

number

[0048] Alternatively, the degree of inhibition may be given by parameters functionally linked to INHBE gene expression, such as the amount of protein encoded by the INHBE gene secreted by cells, the level of plasma lipids, or a reduction in the number of cells exhibiting a particular phenotype. In principle, silencing of the INHBE gene can be determined in any cell expressing the target by constitutive or genomic engineering and by any appropriate assay. However, if a criterion is needed to determine whether a given dsRNA inhibits INHBE gene expression to some extent and is therefore included in the disclosure, the assays provided in the following examples shall serve as such a criterion.

[0049] For example, in certain cases, INHBE gene expression is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% upon administration of the double-stranded oligonucleotide of this disclosure. In some embodiments, the INHBE gene is suppressed by at least about 60%, 70%, or 80% upon administration of the double-stranded oligonucleotide of this disclosure. In some embodiments, the INHBE gene is suppressed by at least about 85%, 90%, or 95% upon administration of the double-stranded oligonucleotide of this disclosure.

[0050] As used herein, in the context of INHBE expression, the terms “treat,” “remedy,” etc., mean the reduction or mitigation of a pathological process mediated by INHBE expression. In the context of this disclosure, to the extent that it relates to any of the other conditions listed below herein (other than pathological processes mediated by INHBE expression), the terms “treat,” “remedy,” etc., mean reducing or mitigating at least one symptom associated with such condition, or slowing or reversing the progression of such condition.

[0051] As used herein, the term “effective dose” means the amount that provides therapeutic benefit in treating, preventing, or managing a pathological process mediated by INHBE expression, or the manifest symptoms of a pathological process mediated by INHBE expression. The specific effective dose can be readily determined by a typical healthcare professional and may vary depending on factors known in the art, such as the type of pathological process mediated by INHBE expression, the patient’s medical history and age, the stage of the pathological process mediated by INHBE expression, and the administration of other antipathological processes mediated by INHBE expression agents.

[0052] As used herein, “pharmaceutical composition” comprises a pharmacologically effective amount of dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutic effective amount,” or simply “effective amount” refers to the amount of RNA effective to produce an intended pharmacological, therapeutic, or prophylactic outcome. 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, then a therapeutically effective amount of drug for the treatment of that disease or disorder is the amount required to produce at least a 25% reduction in that parameter. For example, a therapeutically effective amount of dsRNA targeting INHBE can reduce serum levels of INHBE by at least 25%.

[0053] The term “pharmaceutically acceptable carrier” refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inactive diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inactive diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are generally magnesium stearate, stearic acid, or talc. If necessary, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0054] As used herein, "Tri-GalNAc6" refers to the following structure: [ka]

[0055] As used herein, "L96 ligand" or "L96" refers to the following structure: [ka]

[0056] II. Double-stranded ribonucleic acid (dsRNA) In one aspect of this disclosure, double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the INHBE gene in cells within a subject, such as a mammal (e.g., human). The use of these dsRNA oligonucleotides enables targeted degradation of the mRNA of the corresponding gene (INHBE gene) in mammals.

[0057] In certain embodiments, the dsRNA comprises an antisense strand having a complementarity region that is complementary to at least a portion of the mRNA or mRNA fragment formed in the expression of the INHBE gene. In some embodiments, the dsRNA contains at least 70% complementarity to the mRNA or fragment mRNA of human INHBE mRNA.

[0058] In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, each 15 to 30 nucleotides long, and the antisense strand contains at least 15 consecutive nucleotides of the antisense strand sequences shown in Tables 1 and 2. In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, each 15 to 30 nucleotides long, and the antisense strand contains a sequence that is at least 70% or 80% identical to the antisense strand sequences shown in Tables 1 and 2. In some embodiments, the dsRNAs are compounds 100494, 100506, 100509, 100535, 100557, 100561, 100563, 100563, 100569, 100580, 100589, 100604, 100613, 100625, and 100629. In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, each 15 to 30 nucleotides long, and the antisense strand contains at least 15 consecutive nucleotides of the antisense strand sequence shown in Tables 6 and 7. In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, each 15 to 30 nucleotides long, and the antisense strand contains a sequence that is at least 70% or 80% identical to the antisense strand sequence shown in Tables 6 and 7. In some embodiments, the dsRNAs are compounds 100635, 100636, 100637, 100638, 100639, 100640, 100641, 100642, 100643, 100644, 100645, 100646, and 100647. In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, each 15 to 30 nucleotides long, and the antisense strand contains at least 15 consecutive nucleotides of the antisense strand sequences shown in Tables 9 and 10. In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, each 15 to 30 nucleotides long, and the antisense strand contains a sequence that is at least 70% or 80% identical to the antisense strand sequences shown in Tables 9 and 10.In some embodiments, the dsRNAs are compounds 100643, 100647, 100648, 100649, 100650, 100651, 100652, 100653, 100654, 100655, 100656, and 100657. In some embodiments, the INHBE is human INHBE. In some embodiments, the INHBE is human INHBE containing the sequence shown in SEQ ID NO: 588 (NM_031479.5).

[0059] In some embodiments, the sense strand is 70%, 80%, 90%, 95%, or more identical to the sense strands listed in Tables 1 and 2. In some embodiments, the sense strand is 70%, 80%, 90%, 95%, or more identical to the sense strands listed in Tables 6 and 7. In some embodiments, the sense strand contains at least 15 consecutive nucleotides of the sense strand sequences shown in Tables 6 and 7. In some embodiments, the sense strand is 70%, 80%, 90%, 95%, or more identical to the sense strands listed in Tables 9 and 10. In some embodiments, the sense strand contains at least 15 consecutive nucleotides of the sense strand sequences shown in Tables 9 and 10. In some embodiments, the sense strand contains at least 15 consecutive nucleotides of the sense strand sequences shown in Tables 1 and 2. In some embodiments, the sense strand contains at least 16, 17, 18, 19, 20, or 21 consecutive nucleotides of the sense strand sequences shown in Tables 1 and 2. In some embodiments, the sense strand comprises 21 consecutive nucleotides from the sense strand sequences shown in Tables 1 and 2. In some embodiments, the sense strand is selected from the sense strand sequences shown in Tables 1 and 2. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides from the sense strand sequences shown in Tables 6 and 7. In some embodiments, the sense strand comprises 21 consecutive nucleotides from the sense strand sequences shown in Tables 6 and 7. In some embodiments, the sense strand comprises 22 consecutive nucleotides from the sense strand sequences shown in Tables 6 and 7. In some embodiments, the sense strand comprises 23 consecutive nucleotides from the sense strand sequences shown in Tables 6 and 7. In some embodiments, the sense strand is selected from the sense strand sequences shown in Tables 6 and 7. In some embodiments, the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides from the sense strand sequences shown in Tables 9 and 10. In some embodiments, the sense strand comprises 21 consecutive nucleotides of the sense strand sequence shown in Tables 9 and 10. In some embodiments, the sense strand comprises 22 consecutive nucleotides of the sense strand sequence shown in Tables 9 and 10.In some embodiments, the sense strand comprises 23 consecutive nucleotides of the sense strand sequences shown in Tables 9 and 10. In some embodiments, the sense strand sequence is selected from the sense strand sequences shown in Tables 8 and 9.

[0060] In some embodiments, the antisense chain is 70%, 80%, 90%, 95%, or more identical to the antisense chains listed in Tables 1 and 2. In some embodiments, the antisense chain contains at least 16, 17, 18, 19, 20, or 21 consecutive nucleotides of the antisense chain sequences shown in Tables 1 and 2. In some embodiments, the antisense chain contains 21 consecutive nucleotides of the antisense chain sequences shown in Tables 1 and 2. In some embodiments, the antisense chain is selected from the antisense chain sequences shown in Tables 1 and 2. In some embodiments, the antisense chain is 70%, 80%, 90%, 95%, or more identical to the antisense chains listed in Tables 6 and 7. In some embodiments, the antisense chain contains at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of the antisense chain sequences shown in Tables 6 and 7. In some embodiments, the antisense chain comprises 21 consecutive nucleotides from the antisense chain sequences shown in Tables 6 and 7. In some embodiments, the antisense chain comprises 22 consecutive nucleotides from the antisense chain sequences shown in Tables 6 and 7. In some embodiments, the antisense chain comprises 23 consecutive nucleotides from the antisense chain sequences shown in Tables 6 and 7. In some embodiments, the antisense chain is selected from the antisense chain sequences shown in Tables 6 and 7. In some embodiments, the antisense chain is 70%, 80%, 90%, 95%, or more identical to the antisense chains listed in Tables 9 and 10. In some embodiments, the antisense chain comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides from the antisense chain sequences shown in Tables 9 and 10. In some embodiments, the antisense chain comprises 21 consecutive nucleotides from the antisense chain sequences shown in Tables 9 and 10. In some embodiments, the antisense strand comprises 22 consecutive nucleotides of the antisense strand sequence shown in Tables 9 and 10. In some embodiments, the antisense strand comprises 23 consecutive nucleotides of the antisense strand sequence shown in Tables 9 and 10.In some embodiments, the antisense chain is selected from the antisense chain sequences shown in Tables 9 and 10.

[0061] In some embodiments, the sense chain array is selected from the sense chain arrays shown in Tables 1 and 2, and the antisense chain is selected from the antisense chain arrays shown in Tables 1 and 2. In some embodiments, the sense chain array is selected from the sense chain arrays shown in Tables 6 and 7, and the antisense chain is selected from the antisense chain arrays shown in Tables 6 and 7. In some embodiments, the sense chain array is selected from the sense chain arrays shown in Tables 9 and 10, and the antisense chain is selected from the antisense chain arrays shown in Tables 9 and 10.

[0062] In some embodiments, the dsRNA has a mismatch with respect to the INHBE mRNA fragment. In some embodiments, the dsRNA contains one or two mismatches with respect 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. In some embodiments, the antisense strand contains a nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the target mRNA corresponding to the INHBE mRNA fragment. In some embodiments, the antisense strand of the dsRNA contains at least 80% complementarity with respect to the INHBE mRNA fragment. In some embodiments, the mismatch is located in the sense strand. In some embodiments, the mismatch is located in the antisense strand. In some embodiments, the antisense strand of the dsRNA contains one, two, three, or four mismatches with respect to the INHBE mRNA fragment. In some embodiments, the mismatches are located in the center of the dsRNA. In some embodiments, the mismatches are located in the 5' or 3' region of the dsRNA. In some embodiments, the mismatches are within 5 nucleotides from the 5' or 3' end of the dsRNA.

[0063] In some embodiments, at least one strand of the dsRNA contains a 3' or 5' overhang of at least one nucleotide. In some embodiments, the overhang is at least two nucleotides or at least three nucleotides. In some embodiments, at least one strand of the dsRNA contains a 3' overhang. In some embodiments, at least one strand of the dsRNA contains a 5' overhang.

[0064] In some embodiments, the antisense strand has a 3' terminal nucleotide overhang compared to the sense strand. In some embodiments, the 3' terminal nucleotide overhang contains one, two, or three nucleotides compared to the sense strand. In some embodiments, the antisense strand 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 contain at least one, at least two, at least three, or at least four mismatched nucleotides.

[0065] dsRNA can be synthesized by standard methods known in the art, as will be discussed further below, for example, by using automated DNA synthesizers commercially available from Biosearch, Applied Biosystems, Inc. dsRNA contains two RNA strands that are sufficiently complementary to hybridize to form a double-stranded structure. One strand of dsRNA (the antisense strand) contains a complementary region that is complementary to a target sequence derived from the mRNA sequence formed during INHBE gene expression, and the other strand (the sense strand) contains a region that is complementary to the antisense strand such that, when combined under favorable conditions, the two strands hybridize to form a double-stranded structure.

[0066] In some embodiments, the double-stranded structure is 15–30, 25–30, 18–25, 19–24, 19–21, or 19, 20, or 21 base pairs long. In one embodiment, the double-stranded structure is 19 base pairs long. In another embodiment, the double-stranded structure is 20 base pairs long. In yet another embodiment, the double-stranded structure is 21 base pairs long. When two different single-stranded RNAs (ssRNAs) are used in combination, the lengths of the double-stranded structures may be the same or different.

[0067] In some embodiments, each strand of the dsRNA of this disclosure is 15–30, or 18–25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. In other embodiments, each strand is approximately 25–30 nucleotides long. In some embodiments, each strand of the double helix is ​​the same length or of different lengths. When two different ssRNAs are used in combination, the lengths of each strand of each ssRNA may be the same or different.

[0068] In some embodiments, the dsRNA is longer than 21–23 nucleotides, and includes, for example, a dsRNA long enough to be processed into 21–23 base pair siRNAs by the RNase III enzyme Dicer, which is then incorporated into the RNA-induced silencing complex (RISC). Thus, the dsRNAs of this disclosure are at least 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or at least 100 base pairs long.

[0069] Inhibition of INHBE gene expression can be assayed, for example, by nucleic acid-based assays such as quantitative PCR, or by protein-based methods such as Western blotting. INHBE gene expression can be reduced by at least 50% when measured by the assays described in the following examples. For example, INHBE gene expression 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.

[0070] In another aspect, the disclosure provides single-stranded antisense oligonucleotides (RNAi). An antisense oligonucleotide is a single-stranded oligonucleotide complementary to a sequence within a target mRNA. Antisense oligonucleotides can inhibit translation in a stoichiometric manner by physically interfering with base pairing and translation mechanisms to mRNA. See Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355. Antisense oligonucleotides can also inhibit target protein expression by binding to an mRNA target and promoting mRNA target disruption via ribonuclease H (RNase-H). Single-stranded antisense RNA molecules can be approximately 13 to 30 nucleotides in length and have a sequence complementary to the target sequence. For example, a single-stranded antisense RNA molecule may contain a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one of the antisense sequences in Tables 1 and 2, Tables 6 and 7, or Tables 9 and 10.

[0071] qualification In certain embodiments, dsRNAs are chemically modified to enhance their stability. The nucleic acids characterized in this disclosure may be synthesized and / or modified by methods established in the art, such as those described in, for example, "Current protocols in nucleic acid chemistry," Beaucage, S. Let al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Specific examples of dsRNA compounds useful in this disclosure include dsRNAs containing a modified skeleton or unnatural nucleoside-linked bonds. As defined herein, dsRNAs having a modified skeleton include dsRNAs that retain a phosphorus atom in the skeleton and dsRNAs that do not have a phosphorus atom in the skeleton. For the purposes of this disclosure and as may be referred to in the art, modified dsRNAs that do not have a phosphorus atom in their nucleoside-linked skeleton are also oligonucleotides. It may be considered a creoside. In some embodiments, the modified dsRNA backbone comprises at least one of the following: a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a 2'-fluoro modified nucleotide, an inverted base-dropped nucleotide, a thymidine-glycol nucleic acid (GNA) S-isomer, inosine, and an inverted deoxyribonucleotide (3'-3' linked nucleotide or 5'-5' linked nucleotide), a thymidine-glycol nucleic acid (GNA) S-isomer, a nucleotide containing a modified nucleotide component represented by the following formula (I), [ka] and nucleotides comprising a modified nucleotide component represented by the following formula (II): [ka] During the ceremony, B 1 and B 2 Each of these is a nucleic acid base, R 1 is hydrogen and C 1-6 Selected from the group consisting of alkyl groups, The antisense strand and sense strand may optionally each contain at least one modified nucleotide.

[0072] In some embodiments, the nucleotide containing the modified nucleotide component represented by formula (I) is B 1 It contains nucleic acid bases represented by B 1 These are independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and their modified analogs. In some embodiments, the nucleotides containing the modified nucleotide component represented by formula (II) are B 2 It contains nucleic acid bases represented by B 2 In some embodiments, B is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and their modified analogs. 1 and B 2 Each is independently selected from adenine, uracil, cytosine, and their modified analogs. In some embodiments, R 1 C 1-6 It is alkyl. In some embodiments, R 1 is -CH3. In some embodiments, B 1 is uracil. In some embodiments, R 1 It is -CH3, and B 1 is uracil. In some embodiments, B 2 is adenine. In some embodiments, B 2 That is Uracil.

[0073] In some embodiments, the sense strand includes an inverted deoxyribonucleotide at its 3' end, optionally a 3'-3' deoxythymidine. In some embodiments, the sense strand includes an inverted deoxyribonucleotide at its 5' end and an inverted deoxyribonucleotide at its 3' end, optionally a 5'-5' deoxythymidine and a 3'-3' deoxythymidine. In some embodiments, the sense strand includes a nucleotide at its 3' end containing a modified nucleotide component represented by formula (I), optionally R in the formula. 1 It is -CH3, and B 1 That is Uracil.

[0074] In some embodiments, modifications include one or more phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphates having the usual 3'-5' linkage, inverted polarity, where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2', and their 2'-5' linked analogues. Various salts, mixed salts, and free acid forms are also included.

[0075] In some embodiments, the modified nucleotide comprises at least one of 5'-vinylphosphonate nucleotide, 5'-phosphate or phosphate mimetic, locked nucleic acid (LNA), 2'-MOE (methoxyethyl) nucleotide, and / or 2'-arabinofluoro (2'-araF) nucleotide. In some embodiments, the antisense chain of the modified nucleotide comprises a phosphate mimetic at the 5' end, optionally being 5'-E-vinyl-phosphonate or 4'-O-phosphonate.

[0076] In some embodiments, the modified nucleotide includes at least one of the following nucleotides: 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy modified nucleotide, locked nucleotide, base-dropped nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, and / or non-natural base.

[0077] In some embodiments, the antisense and / or sense strand includes at least one internucleoside bond selected from the group consisting of phosphorothioate bonds, phosphorodithioate bonds, phosphotryester bonds, alkylphosphonate bonds, aminoalkylphosphotryester bonds, alkylenephosphonate bonds, phosphine bonds, phosphoramidate bonds, phosphormololate bonds, phosphoropiperazidate bonds, aminoalkylphosphoamidate bonds, thiophosphoamidate bonds, thionoalkylphosphonate bonds, thionoalkylphosphotryester bonds, thiophosphate bonds, selenophosphate bonds, and boranophosphate bonds. In some embodiments, the antisense and / or sense strand includes at least one nucleotide modification bond. In some embodiments, all nucleotide bonds in the antisense strand are modification bonds. In some embodiments, the antisense and / or sense strand includes at least one phosphorothioate (PS) bond.

[0078] Conjugate

[0079] Another modification of the dsRNAs of this disclosure includes chemically linking one or more ligands or targeting moieties or conjugates (e.g., GalNAc of this disclosure, e.g., tri-GalNAc6) to the dsRNAs that enhance the activity, cell distribution, or cell uptake of the dsRNAs, such moieties include cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306-309, Manoharan et al.) al., Biorg. Med. Chem. Let., 1993, 3:2765-2770, thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains, e.g., dodecanediol residues or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118, Kabanov et al., FEBS Lett., 1990, 259:327-330, Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654, Shea et al., Nucl. Acids Res., 1990, 18:3777-3783, polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys.Examples include, but are not limited to, lipid moieties such as Acta, 1995, 1264:229-237, or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0080] In some embodiments, the ligand or targeting moiety (e.g., GalNAc as disclosed, e.g., tri-GalNAc6) is conjugated to the 5' end, 3' end, or both ends of the dsRNA. In some embodiments, the ligand or targeting moiety (e.g., GalNAc as disclosed, e.g., tri-GalNAc6) is conjugated to the 3' end of the sense strand of the dsRNA. In some embodiments, the ligand or targeting moiety (e.g., GalNAc as disclosed, e.g., tri-GalNAc6) is conjugated to the 3' end of the antisense strand of the modified dsRNA. In some embodiments, the ligand or targeting moiety (e.g., GalNAc as disclosed, e.g., tri-GalNAc6) is conjugated to the 5' end of the sense strand of the dsRNA. In some embodiments, the ligand or targeting moiety (e.g., GalNAc as disclosed herein, e.g., tri-GalNAc6) is conjugated to the 5' end of the antisense strand of the modified dsRNA. In some embodiments, the ligand or targeting moiety is at least one N-acetyl-galactosamine (GalNAc).

[0081] In some embodiments, dsRNA can be modified with non-ligand groups. Numerous non-ligand molecules have been conjugated to dsRNA to enhance its activity, cell distribution, or cell uptake, and procedures for carrying out such conjugations are available in the scientific literature.Examples of such non-ligand moieties include cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYAcad. Sci., 1992, 660:306, Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains, such as dodecanediol residues or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111, Kabanov et al., FEBS Lett., 1990, 259:327, Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651, Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetate (Manoharan et al., Tetrahedron Examples include lipid moieties such as Lett., 1995, 36:3651, palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Typical conjugation protocols involve the synthesis of dsRNAs that hold aminolinkers at one or more positions in the oligonucleotide sequence.Next, the amino group is reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out either using the dsRNA still bound to the solid support or after cleavage of the dsRNA in the solution phase. The dsRNA conjugate can be purified, for example, by HPLC.

[0082] Conjugating ligands to dsRNAs allows for targeting specific tissues, enhancing uptake by specific cell types such as hepatocytes, as well as cellular absorption. In certain cases, hydrophobic ligands, when conjugated to dsRNAs, promote direct transmembrane penetration and / or transcatheter uptake across hepatocytes. Alternatively, ligands conjugated to dsRNAs are substrates for receptor-mediated endocytosis. These approaches have been used to facilitate the cellular penetration of antisense oligonucleotides and dsRNA reagents. For example, cholesterol can be conjugated to various antisense oligonucleotides, resulting in substantially more active compounds compared to their unconjugated analogs. See M. Manoharan, Antisense & Nucleic Acid Drug Development 2002,12,103. Other lipophilic compounds conjugated to oligonucleotides include 1-pyrenebutyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. Folic acid is an example of a ligand for receptor-mediated endocytosis. Folic acid enters cells via folate receptor-mediated endocytosis. dsRNA compounds that hold folic acid are efficiently transported into cells via folate receptor-mediated endocytosis. Li and collaborators reported that adding folic acid to the 3'-terminus of oligonucleotides resulted in an eightfold increase in the cellular uptake of the oligonucleotides. Li, S.; Deshmukh, HM; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands conjugated to oligonucleotides include polyethylene glycol, carbohydrate clusters, crosslinkers, porphyrin conjugates, delivery peptides, and lipids such as cholesterol and cholesterylamines. Examples of carbohydrate clusters include Chol-p-(GalNAc)3 (N-acetylgalactosamine cholesterol) and LCO(GalNAc)3 (N-acetylgalactosamine-3'-lithochol-oleoyl).

[0083] Carbohydrate conjugate In some embodiments, the dsRNA oligonucleotides of this disclosure further contain carbohydrates. Carbohydrate-conjugated dsRNAs are advantageous for the in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, “carbohydrate” means either a carbohydrate itself, which consists of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound which has as part a carbohydrate portion consisting of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Examples of specific monosaccharides include sugars with C5 or more (e.g., C5, C6, C7, or C8), while examples of disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0084] In some embodiments, the carbohydrate conjugate for use in the compositions and methods of the present disclosure is a monosaccharide. In some embodiments, the monosaccharide is a monosaccharide of the following formula I or formula II [ka] It is N-acetylgalactosamine.

[0085] In some embodiments, the carbohydrate is conjugated to the 5' end, 3' end, or both ends of the modified dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3' end of the sense strand of the modified dsRNA. In some embodiments, the ligand or targeting moiety is conjugated to the 3' end of the antisense strand of the modified dsRNA. In some embodiments, the carbohydrate is at least one N-acetyl-galactosamine (GalNAc).

[0086] Mai. Pharmaceutical composition This specification also discloses pharmaceutical compositions comprising dsRNA that target the INHBE gene of this disclosure.

[0087] In certain embodiments, this disclosure provides pharmaceutical compositions containing the dsRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing dsRNA are useful for treating diseases or disorders associated with the expression or activity of a targeting INHBE gene, such as pathological processes mediated by the expression of a targeting INHBE gene. Such pharmaceutical compositions are formulated based on a delivery mode.

[0088] The pharmaceutical compositions described herein are administered in a dose sufficient to inhibit the expression of the INHBE gene.

[0089] Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and other pre-existing conditions, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective dose of a composition may consist of a single treatment or a series of treatments. Estimations of the effective dose and in vivo half-life of individual dsRNAs contained herein can be made using conventional methodologies or based on in vivo studies using appropriate animal models, as described elsewhere in this specification.

[0090] Advances in mouse genetics have led to the development of numerous mouse models for studying various human diseases, including pathological processes mediated by INHBE expression. Such models are used not only for in vivo testing of dsRNAs but also for determining effective therapeutic doses. 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 expressing human INHBE.

[0091] Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in humans. Doses of the compositions featured in this disclosure generally fall within a range of blood concentrations, including ED50, that are little to no toxicity. This dose may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods featured in this disclosure, the therapeutically effective dose can be initially estimated from cell culture assays. If the dose is determined in cell culture, it can be determined in an animal model to achieve a circulating plasma concentration range of the target sequence compound, including IC50 (i.e., the concentration of the test compound that achieves half-symptom inhibition), or, where appropriate, a circulating plasma concentration range of the target sequence polypeptide product (e.g., to achieve a reduction in polypeptide concentration). This information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0092] The dsRNAs featured in this disclosure can be administered in combination with other known reagents effective in treating pathological processes mediated by INHBE gene expression. In any case, the administering physician may adjust the amount and timing of dsRNA administration based on results observed using standard measures of efficacy known in the art or described herein.

[0093] Liposome formulations In certain embodiments, the pharmaceutical compositions disclosed herein include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing pharmaceutical compositions that include hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0094] In some embodiments, the dsRNA of this disclosure is introduced into a pre-formed liposome or lipoplex made from a mixture of cationic and neutral lipids. In certain methods, dsRNA complexes having monocationic or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, the lipid portion is selected to increase the distribution of the drug to specific cells or tissues. In certain embodiments, the lipid portion is selected to increase the distribution of the drug to adipose tissue. In certain embodiments, the lipid portion is selected to increase the distribution of the drug to muscle tissue.

[0095] Excipients In certain embodiments, the pharmaceutical composition includes an excipient. In contrast to a carrier compound, the “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspension, 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 mode of administration in mind so as to provide the desired volume, homogeneity, etc., when combined with the nucleic acids and other components of the given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate), disintegrants (e.g., starch, sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate).

[0096] The compositions of this disclosure may also be formulated using pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not react adversely with nucleic acids. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0097] Nucleic acid topical formulations may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of nucleic acids in liquid or solid oily bases. The solutions may also contain buffers, diluents, and other suitable additives. Pharmacochemically acceptable organic or inorganic excipients suitable for parenteral administration that do not react adversely with nucleic acids may be used.

[0098] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0099] Other components The compositions of this disclosure may additionally include other auxiliary components conventionally found in pharmaceutical compositions, at levels of use established in the art. For example, a composition may contain additional pharmaceutically active materials of additional suitability, such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents, or additional materials useful for physically formulating various dosage forms of the compositions of this disclosure, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not excessively interfere with the biological activity of the components of the compositions of this disclosure. The formulations can be sterilized and, if desired, can be mixed with auxiliary agents that do not have harmful interactions with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavorings, and / or aromatics.

[0100] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0101] Administration This specification also discloses methods for administering pharmaceutical compositions and formulations, including the dsRNA compositions and pharmaceutical compositions of this disclosure. In some embodiments, the dsRNA compositions or pharmaceutical compositions of this disclosure are administered in multiple ways, depending on whether topical or systemic treatment is preferred and the area to be treated.

[0102] In certain embodiments, administration of the pharmaceutical composition may be topical (including buccal and sublingual), such as by inhalation or insufflation of powder or aerosol into the lungs, including by nebulizer, intratracheal, intranasal, epidermal and transdermal, or oral or parenteral. In some embodiments, parenteral administration may be by intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion, or intracranial, such as intraparenchymal, subarachnoid, or intraventricular administration.

[0103] The pharmaceutical compositions containing the dsRNA of this disclosure can be presented in unit dosage forms and can be prepared by any preferred method. The pharmaceutical compositions should be formulated to suit their intended route of administration. Useful formulations can be prepared by methods well known in the pharmaceutical art. See, for example, Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).

[0104] Pharmaceutical preparations are, for example, sterilized. Sterilization can be achieved, for example, by filtration through a sterile filtration membrane. If the composition is freeze-dried, filter sterilization can be performed before or after freeze-drying and reconstitution.

[0105] In certain embodiments, dsRNA is delivered in a manner that targets a specific tissue, such as the liver.

[0106] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form is the amount of the compound that produces the therapeutic effect (e.g., a dsRNA molecule).

[0107] In certain embodiments, the formulations of the present disclosure comprise an excipient selected from the group consisting of cyclodextrin, cellulose, liposomes, micellar-forming agents such as bile acids, and polymer carriers such as polyesters and polyanhydrides, and a compound of the present disclosure (e.g., a dsRNA molecule). In certain embodiments, the aforementioned formulations make the compound of the present disclosure (e.g., a dsRNA molecule) orally bioavailable.

[0108] Formulations of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (usually using a flavored base which is sucrose and acacia or tragacanth), powders, granules, or as a solution or 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 a lozenge (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of the compound of the present disclosure (e.g., a dsRNA molecule) as an active ingredient. The compound of the present disclosure (e.g., a dsRNA molecule) may also be administered as a bolus, lick, or paste.

[0109] Liquid dosage forms for oral administration of the compounds of this disclosure (e.g., dsRNA molecules) include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.

[0110] IV. Methods for inhibiting INHBE gene expression In one embodiment, the disclosure provides a method for inhibiting the expression of an INHBE gene in a cell. The method comprises administering a dsRNA targeting the INHBE gene to a cell so as to reduce the expression of the target INHBE gene in the cell. The disclosure includes methods carried out in cells in vitro or in vivo. In some embodiments, the methods are carried out in animal cells, such as mouse, rat, non-human primate, or human cells.

[0111] The Disclosure also provides a method for using the dsRNA of the Disclosure and / or a composition containing the dsRNA of the Disclosure to reduce and / or inhibit intracellular INHBE expression. The method comprises contacting cells with the dsRNA of the Disclosure and maintaining the cells for a time sufficient to obtain degradation of the mRNA transcript of the INHBE gene, thereby inhibiting intracellular INHBE gene expression. The reduction in gene expression can be assessed by any method known in the Art. For example, the reduction in INHBE expression can be determined by determining the mRNA expression level of INHBE using methods common to those skilled in the art, such as Northern blotting, qRT-PCR; by determining the protein level of INHBE using methods common to those skilled in the art, such as Western blotting, immunological techniques; and / or by determining the biological activity of INHBE, which is, for example, affecting one or more molecules related to the cellular blood coagulation mechanism (or blood coagulation itself in an in vivo environment).

[0112] In the methods of this disclosure, cells may be brought into contact in vitro or in vivo, i.e., cells may be within the object.

[0113] Cells suitable for treatment using the methods of this disclosure may be any cells expressing the INHBE gene. Cells suitable for use in the methods of this disclosure may be mammalian cells, such as primate cells (human cells, or non-human primate cells such as monkey cells or chimpanzee cells), non-primate cells (such as bovine cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), avian cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cells are human cells, such as human hepatocytes.

[0114] In some embodiments, INHBE expression is inhibited by at least 30% compared to a control after administration of the dsRNA oligonucleotide of this disclosure.

[0115] In some embodiments, a method is provided for treating an INHBE-mediated disorder, which includes administering a therapeutically effective amount of the dsRNA oligonucleotide or pharmaceutical composition of the Disclosure to a subject in need of such treatment.

[0116] In some embodiments, the disorder is a cardiovascular disorder. In some embodiments, the disorder is a cardiovascular disease. [Examples]

[0117] The following embodiments are provided for the purpose of illustrating various embodiments of the Disclosure and are not intended to limit the Disclosure in any way. These embodiments, together with the methods described herein, represent currently preferred embodiments, are illustrative, and are not intended as limitations on the scope of the Disclosure. Any modifications and other uses thereof that are incorporated within the spirit of the Disclosure as defined by the claims will be conceivable to those skilled in the art.

[0118] Example 1. In vitro RNA interference (RNAi) screening in Huh-7 cell line This example describes the screening of INHBE gene inhibition by siRNA in a hepatocyte-derived cancer cell line model (Huh-7). Briefly, Huh-7 cells were transfected with 147 3'-GalNAc-conjugated modified siRNAs (double-stranded 100488-100634, sense strands of SEQ ID NOs. 294-440 and antisense strands of SEQ ID NOs. 441-587) at 10 nM and 0.1 nM concentrations. Exemplary sequences of unmodified and modified siRNA compounds are shown in Tables 1 and 2, respectively. The compounds in Table 2 were 3'GalNAc-modified. INHBE mRNA levels were measured by quantitative PCR and normalized to GAPDH compared to mock-treated control cells. Table 3 and Figures 1A, B, and C show the results of single-dose screening at 10 nM and 0.1 nM in Huh7 cells using selected INHBE siRNAs. The data are presented as the inhibition rate of INHBE mRNA in siRNA-transfected cells compared to INHBE mRNA in mock-treated control cells.

[0119] [Table 1-1]

[0120] [Table 1-2]

[0121] [Table 1-3]

[0122] [Table 1-4]

[0123] [Table 2-1]

[0124] [Table 2-2]

[0125] [Table 2-3]

[0126] [Table 2-4]

[0127] [Table 3-1]

[0128] [Table 3-2]

[0129] [Table 3-3]

[0130] [Table 3-4]

[0131] Example 2. In vitro dose-response screening in the Huh7 cell line This example describes the screening of exemplary INHBE siRNA compounds in primary human hepatocytes (PHH) cells in single-dose screening at 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.412 nM, 0.137 nM, and 0.046 nM of selected siRNAs (Table 2). INHBE mRNA levels were measured by quantitative PCR, normalized to GAPDH compared to mock-treated control cells, and mean KD and SD were determined. Data are presented as the percentage inhibition rate of INHBE mRNA in siRNA-treated cells compared to INHBE mRNA in PBS control cells. Table 4 and Figure 2 show the results of dose-response INHBE siRNA screening in vitro.

[0132] [Table 4]

[0133] Example 3. In vitro dose-response screening in primary human hepatocytes This example describes the screening of exemplary INHBE siRNA compounds in primary human hepatocytes (PHH) cells in single-dose screening of selected siRNAs at 10 nM and 1 nM (Table 2). INHBE mRNA levels were measured by quantitative PCR, normalized to GAPDH compared to mock-treated control cells, and mean KD and SD were determined. Data are presented as the percentage inhibition rate of INHBE mRNA in siRNA-treated cells compared to INHBE mRNA in PBS control cells. Table 5 and Figure 3 show the results of dose-response INHBE siRNA screening in vitro.

[0134] [Table 5]

[0135] Example 4. Evaluation of human INHBE knockdown using selected siRNA in an in vivo mouse hydrodynamic injection (HDI) model. Thirteen exemplary siRNA compounds (compounds 100635–100647) were tested for knockdown of human INHBE in a mouse model hydrodynamically injected with a DNA plasmid encoding a full-length human INHBE transcript. Briefly, 6–7-week-old female BALB / c mice were subcutaneously injected with 1 mg / kg of the INHBE siRNA compounds from Table 6. Three days after injection, the mice were hydrodynamically injected with a DNA plasmid encoding a full-length human INHBE transcript. One day after plasmid injection, liver samples were collected and analyzed for INHBE mRNA expression compared to mice treated with the same amount of PBS. INHBE mRNA levels were measured by quantitative PCR and normalized to the NEO gene contained in the plasmid used to express INHBE. Data are presented as relative gene expression of INHBE mRNA in the liver compared to PBS-treated animals.

[0136] The modified and unmodified sense and antisense chain sequences of compounds 100329-100341 are summarized in Tables 6-7.

[0137] Table 8 and Figure 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%. Furthermore, compounds 100635–100646 all showed improved efficacy compared to siRNA compound 100647.

[0138] [Table 6-1]

[0139] [Table 6-2]

[0140] [Table 7]

[0141] [Table 8]

[0142] Example 5. Evaluation of human INHBE knockdown using selected siRNA in an in vivo mouse hydrodynamic injection (HDI) model. Twelve exemplary siRNA compounds (compounds 100643 and 100647-100657) were tested for knockdown of human INHBE in a mouse model hydrodynamically injected with a DNA plasmid encoding a full-length human INHBE transcript. Briefly, 6-7 week old female BALB / c mice were subcutaneously injected with 1 or 1.5 mg / kg of INHBE siRNA compounds from Table 9. Three days after injection, the mice were hydrodynamically injected with a DNA plasmid encoding a full-length human INHBE transcript. One day after plasmid injection, liver samples were collected and analyzed for INHBE mRNA expression compared to mice treated with the same amount of PBS. INHBE mRNA levels were measured by quantitative PCR and normalized to the NEO gene contained in the plasmid used to express INHBE. Data are presented as relative gene expression of INHBE mRNA in the liver compared to PBS-treated animals.

[0143] The modified and unmodified sense and antisense chain sequences of compounds 100643 and 100647-100657 are summarized in Tables 9-10.

[0144] Figure 5 shows the results of single-dose INHBE siRNA injection in INHBE BALB / c mice. The results indicate that siRNA compounds 100643, 100649, 100650, 100654, 100655, and 100657 showed improved efficacy compared to siRNA compound 100647.

[0145] [Table 9-1]

[0146] [Table 9-2]

[0147] [Table 10]

[0148] Example 6. Evaluation of INHBE knockdown using siRNA in a non-human primate model in vivo. Two exemplary siRNA compounds, referred to herein as Compound A and Compound B, were selected from the compounds listed in Tables 6 and 9, and tested for INHBE knockdown in non-human primate models. Briefly, male cynomolgus monkeys aged 3–8 years were subcutaneously injected with either INHBE siRNA compound A or B at a dose of 5 mg / kg (n=2–3 monkeys per test compound). The animals were clinically observed twice daily after injection. Liver biopsies were taken 4 days before injection and 56 days after injection. Liver INHBE mRNA levels were measured by quantitative PCR and normalized to the level at day 4 for each individual animal.

[0149] Figure 6 shows the results of single-dose INHBE siRNA injection in cynomolgus monkeys. The results show that 69% knockdown (compound A) and 75% knockdown (compound B) were observed in the animals 56 days after injection (Figure 6).

[0150] Example 7. In vitro activity and dose-response screening in hTLR7, hTLR8, and hTLR9 cells. This example evaluates the agonist activity of compounds in cell-based human TLR Toll-like receptor (hTLR) reporter assays. Exemplary siRNA compounds 100647, 100635, 100638, 100639, 100642, 100643, and 10064 were tested using commercially available cell-based hTLR7, hTLR8, and hTLR9 reporter assays. Briefly, the assays were performed using 4-fold dilutions from 100 nM at nine concentrations with a 24-hour treatment period by transfecting HEK-293 cells in pairs. R848 was purchased from a commercial vendor and used as an agonist in the hTLR7 and hTLR8 reporter assays. ODN 2006 was purchased from a commercial vendor and used as an agonist in the hTLR9 reporter assay.

[0151] The data are presented as the fold change in activity relative to unstimulated cells in cell-based hTLR7, hTLR8, and hTLR9 reporter assays, and as the activity level in cells treated with the test compound. Table 11 and Figures 7A–7C show the results of the cell-based hTLR7, hTLR8, and hTLR9 reporter assays. The results indicate that none of the test siRNA compounds exhibited activity against the hTLR7, hTLR8, and hTLR9 pathways.

[0152] [Table 11]

[0153] Example 8. RNA sequence transcriptome analysis in primary human hepatocytes This example evaluates the RNA sequence transcriptome in primary human hepatocytes to assess the potential off-target risks of exemplary siRNA compounds 100647, 100635, 100638, 100639, 100642, 100643, and 100645. Briefly, primary human hepatocytes (PHH) were collected 48 hours after treatment with exemplary siRNA compounds for RNA extraction, library construction, and sequencing.

[0154] Figure 8 shows the results of RNA sequence transcriptome analysis in primary human hepatocytes treated with exemplary siRNA compounds. The data are presented as a volcano plot of differentially expressed genes (DEGs) across different groups, showing that INHBE is significantly downregulated in all groups. Of all the exemplary siRNA compounds tested, compound 100639 yielded the most complete RNA-seq results.

[0155] Example 9. Non-GLP small toxicity test of selected siRNA in a mouse model in vivo Exemplary siRNA compounds 100635, 100642, and 100643 were evaluated via a non-GLP miniature toxicity study in mice. Briefly, 7-week-old male C57BL / 6J mice (5 mice per group) were subcutaneously injected with a single dose of 50 mg / kg of one of the siRNA compounds 100635, 100642, or 100643 (i.e., on day 0).

[0156] Blood samples were taken on day 0 (before administration) and day 7. Tissue samples (liver and kidney) were collected 7 days after the end of treatment.

[0157] Urine and blood samples collected on day 0 (before administration) were handled as follows: Plasma was rapidly frozen on dry ice at the time of collection, stored at -80°C, and transferred to biochemical analysis. Urine was stored at 4°C or -80°C until transferred to biochemical analysis.

[0158] Urine, blood, liver, and kidney samples collected on day 7 were handled as follows: Plasma: Rapidly frozen on dry ice at the time of collection, stored at -80°C, and transferred to biochemical analysis. Plasma was stored on ice until transferred to coagulation assays.

[0159] The liver and kidneys were fixed in 4% paraformaldehyde until transferred for histopathological evaluation.

[0160] Figure 9 and Table 12 show the results of injecting C57BL / 6J mice with a single dose of 50 mg / kg of one of the siRNA compounds 100635, 100642, or 100643.

[0161] Figure 9 shows the results of biochemical tests in mice over 7 days after administration. Compared to the PBS control group, no significant differences were observed in the mean plasma concentrations 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), and lactate dehydrogenase (LDH) in the test compound group mice. The mean plasma UREA concentration in the 100635 group mice was significantly higher than the mean plasma UREA concentration in the PBS group mice on day 7. Compared to the PBS control group, no significant differences were observed in the mean urinary concentrations of urea (UREA), urinary total microprotein (UP), and creatinine (CREZ) in the test compound group mice.

[0162] Table 12 shows the results of liver and kidney pathology tests over 7 days after administration. The test results indicate no significant lesions (damage) in the subjects. Changes observed in the PBS group may be due to background lesions.

[0163] [Table 12]

[0164] In conclusion, no significant differences were detected in the biochemical indicators tested, and no significant lesions or damage were observed in the pathological diagnoses of the liver and kidneys. Therefore, overall, subcutaneous administration of a single dose of test compound 100635, 100642, or 100643 in mice was considered safe, and no apparent toxicity was observed in this non-GLP study.

[0165] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that these embodiments are provided merely as examples. Numerous variations, modifications, and substitutions will be conceivable to those skilled in the art without departing from the present invention. Naturally, various alternatives to the embodiments described herein may be employed. The following claims define the scope of the present invention, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be covered thereby.

[0166] [Table 13-1]

[0167] [Table 13-2]

[0168] [Table 13-3]

[0169] [Table 13-4]

[0170] [Table 13-5]

[0171] Table 13-6

[0172] Table 13-7

[0173] Table 13-8

[0174] Table 13-9

[0175] Table 13-10

[0176] Table 13-11

[0177] Table 13-12

[0178] Table 13-13

[0179] Table 13-14

[0180] Table 13-15

[0181] [Table 13-16]

[0182] [Table 13-17]

[0183] Built-in by reference Each of the patent and scientific literature disclosures referred to herein is incorporated by reference for all purposes.

[0184] Equal parts This disclosure can be embodied in other specific forms without departing from its essential features. Therefore, the embodiments described herein should be considered illustrative rather than limiting the disclosure described herein. The scope of this disclosure is indicated by the appended claims rather than the foregoing description, and all modifications that fall within the meaning and scope of equivalence of the claims are intended to be encompassed therein.

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of angiotensinogen (INHBE), wherein the dsRNA comprises a sense strand and an antisense strand, each having a length of 15 to 30 nucleotides. (a) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 598, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 589, (b) The antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 599, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 590, (c) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 600, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No.

591. (d) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 601, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 592, (e) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 602, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 593, (f) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 603, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 594, (g) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 604, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 595, (h) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 605, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 596, or (i) A double-stranded ribonucleic acid (dsRNA) wherein the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 606, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No.

597.

2. A double-stranded ribonucleic acid (dsRNA) for inhibiting INHBE expression, wherein the dsRNA comprises a sense strand and an antisense strand, each having a length of 15 to 30 nucleotides. (a) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 616, and the sense strand includes 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 Sequence ID No. 617, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 608, (c) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 618, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 609, (d) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 619, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 610, (e) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 620, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 611, (f) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 621, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No.

612. (g) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 622, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No.

613. (h) The antisense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 623, and the sense strand includes a sequence that is at least 70% or 80% identical to the sequence of Sequence ID No. 614, or (i) A double-stranded ribonucleic acid (dsRNA) wherein the antisense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO: 624, and the sense strand comprises a sequence that is at least 70% or 80% identical to the sequence of SEQ ID NO:

615.

3. The dsRNA according to claim 1 or claim 2, wherein the INHBE is human INHBE.

4. The dsRNA according to claim 1 or claim 2, wherein the INHBE is a human INHBE containing the sequence shown in Sequence ID No. 588 (NM_031479.5).

5. The dsRNA according to claim 1 or 2, wherein the sense strand is 70%, 80%, 90%, 95%, or more identical to a sense strand sequence containing 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: 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, or SEQ ID NO:

615.

6. The dsRNA according to claim 1 or 2, wherein the sense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of a sense strand sequence including 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: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ 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.

7. The aforementioned sense chain is (a) Twenty consecutive nucleotides of a sense strand sequence containing 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: 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, or SEQ ID NO: 615, (b) 21 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, or SEQ ID NO: 615, (c) 22 consecutive nucleotides of a sense strand sequence containing the sequence of SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 612, SEQ ID NO: 613, SEQ ID NO: 614, or SEQ ID NO: 615, and / or (d) The dsRNA according to claim 1, comprising 23 consecutive nucleotides of a sense strand sequence including the sequence of SEQ ID NO: 594 or SEQ ID NO:

612.

8. The dsRNA according to claim 1, wherein the antisense strand comprises at least 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides of an antisense sense strand sequence including 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, or SEQ ID NO:

624.

9. The aforementioned antisense chain is (a) 21 consecutive nucleotides of an antisense sense chain sequence containing 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, or SEQ ID NO: 624, (b) 22 consecutive nucleotides of an antisense sense strand sequence containing 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, or SEQ ID NO: 624, and / or (c) The dsRNA according to claim 1, comprising 23 consecutive nucleotides of an antisense sense strand sequence including 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, or SEQ ID NO:

624.

10. The dsRNA according to claim 1 or 2, wherein the sense strand sequence is selected from a sense strand sequence including 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: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ 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, and the antisense strand is selected from an antisense strand sequence including 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, or SEQ ID NO:

624.

11. The dsRNA according to claim 1 or claim 2, wherein the sense strand sequence is selected from the sense strand sequences of SEQ ID NO: 589, SEQ ID NO: 590, SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 594, SEQ ID NO: 595, SEQ ID NO: 596, SEQ 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.

12. The dsRNA according to claim 1 or claim 2, wherein the antisense strand is selected from the antisense strand sequences 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.

13. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 598, and the sense strand comprises the sequence of SEQ ID NO:

589.

14. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 599, and the sense strand comprises the sequence of SEQ ID NO:

590.

15. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 600, and the sense strand comprises the sequence of SEQ ID NO:

591.

16. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 601, and the sense strand comprises the sequence of SEQ ID NO:

592.

17. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 602, and the sense strand comprises the sequence of SEQ ID NO:

593.

18. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 603, and the sense strand comprises the sequence of SEQ ID NO:

594.

19. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 604, and the sense strand comprises the sequence of SEQ ID NO:

595.

20. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 605, and the sense strand comprises the sequence of SEQ ID NO:

596.

21. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 606, and the sense strand comprises the sequence of SEQ ID NO:

597.

22. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 616, and the sense strand comprises the sequence of SEQ ID NO:

607.

23. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 617, and the sense strand comprises the sequence of SEQ ID NO:

608.

24. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 618, and the sense strand comprises the sequence of SEQ ID NO:

609.

25. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 619, and the sense strand comprises the sequence of SEQ ID NO:

610.

26. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 620, and the sense strand comprises the sequence of SEQ ID NO:

611.

27. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 621, and the sense strand comprises the sequence of SEQ ID NO:

612.

28. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 622, and the sense strand comprises the sequence of SEQ ID NO:

613.

29. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 623, and the sense strand comprises the sequence of SEQ ID NO:

614.

30. The dsRNA according to claim 11 or claim 12, wherein the antisense strand comprises the sequence of SEQ ID NO: 624, and the sense strand comprises the sequence of SEQ ID NO:

615.

31. At least one nucleotide of the dsRNA is a 5'-vinylphosphonate nucleotide, a 2'-O-methyl modified nucleotide, an inverted deoxyribonucleotide (3'-3' linked nucleotide or 5'-5' linked nucleotide), a nucleotide containing a 5'-phosphorothioate group, a 2'-fluoro-modified nucleotide, or a nucleotide containing a modified nucleotide component represented by the following formula (I). 【Chemistry 1】 and nucleotides comprising a modified nucleotide component represented by the following formula (II): 【Chemistry 2】 In the formula, B １ and B ２ Each of these is a nucleic acid base, R １ is hydrogen and C １－６ A modified nucleotide selected from the group consisting of alkyl groups, a nucleotide selected from the group consisting of alkyl groups, The dsRNA according to any one of claims 13 to 30, wherein the antisense strand and the sense strand each optionally contain at least one modified nucleotide.

32. The dsRNA according to any one of claims 1 to 31, wherein the antisense strand has a 3' terminal nucleotide overhang compared to the sense strand.

33. The dsRNA according to claim 32, wherein the 3' terminal nucleotide overhang comprises 1, 2, or 3 nucleotides compared to the sense strand.

34. The dsRNA according to any one of claims 1 to 33, wherein the antisense strand and the sense strand are at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary.

35. The dsRNA according to any one of claims 1 to 33, wherein the antisense strand and the sense strand are at least 80% complementary.

36. The dsRNA according to any one of claims 1 to 33, wherein the antisense strand and the sense strand each contain at least one, at least two, at least three, or at least four mismatched nucleotides.

37. The dsRNA according to any one of claims 1 to 36, wherein 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.

38. The dsRNA according to any one of claims 1 to 36, wherein the antisense strand of the dsRNA has at least 80% complementarity with the INHBE mRNA fragment.

39. The dsRNA according to any one of claims 1 to 36, wherein the antisense strand of the dsRNA contains one, two, three, or four mismatches with respect to the INHBE mRNA fragment.

40. The dsRNA according to any one of claims 1 to 39, wherein at least one nucleotide of the dsRNA is a modified nucleotide.

41. The modified nucleotides include 2'-O-methyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, 2'-fluoro modified nucleotides, inverted base-dropped nucleotides, thymidine-glycol nucleic acid (GNA) S-isomers, inosine, inverted deoxyribonucleotides (3'-3' linked nucleotides or 5'-5' linked nucleotides), thymidine-glycol nucleic acid (GNA) S-isomers, and nucleotides containing a modified nucleotide component represented by the following formula (I): 【Transformation 3】 and nucleotides comprising a modified nucleotide component represented by the following formula (II): 【Chemistry 4】 During the ceremony, B １ and B ２ Each of these is a nucleic acid base, R １ is hydrogen and C １－６ A modified nucleotide selected from the group consisting of alkyl groups, and at least one modified nucleotide selected from the group consisting of alkyl groups, The dsRNA according to claim 40, wherein the antisense strand and the sense strand each optionally include at least one modified nucleotide and a nucleotide containing a modified nucleotide component represented by formula (II).

42. B １ and B ２ The dsRNA according to claim 41, wherein each of B and B is independently selected from the group consisting of adenine, uracil, thymine, cytosine, guanine, and modified analogs thereof.

43. B １ and B ２ The dsRNA according to claim 41 or 42, wherein each is independently selected from adenine, uracil, cytosine, and their modified analogs.

44. R １ C １－６ The dsRNA according to any one of claims 41 to 43, wherein it is alkyl.

45. R １ is, -CH ３ The dsRNA according to any one of claims 41 to 44.

46. B １ The dsRNA according to any one of claims 41 to 45, wherein the dsRNA is uracil.

47. R １ is, -CH ３ B １ The dsRNA according to any one of claims 41 to 46, wherein is uracil.

48. B ２ The dsRNA according to any one of claims 41 to 45, wherein is adenine.

49. B ２ The dsRNA according to any one of claims 41 to 45, wherein the dsRNA is uracil.

50. The dsRNA according to claim 41, wherein the sense strand contains an inverted deoxyribonucleotide at its 5' end, and optionally the inverted deoxyribonucleotide is a 5'-5' linked deoxythymidine.

51. The dsRNA according to claim 41, wherein the sense strand contains an inverted deoxyribonucleotide at its 3' end, and optionally the inverted deoxyribonucleotide is a 3'-3' linked deoxythymidine.

52. The dsRNA according to claim 41, wherein the sense strand comprises an inverted deoxyribonucleotide at its 5' end and an inverted deoxyribonucleotide at its 3' end, wherein the inverted deoxyribonucleotide at the 5' end is optionally a 5'-5' linked deoxythymidine, and the inverted deoxyribonucleotide at the 3' end is a 3'-3' linked deoxythymidine.

53. The sense strand contains a nucleotide with a modified nucleotide component represented by formula (I) at its 3' end, and optionally R in the formula. １ is, -CH ３ B １ The dsRNA according to claims 41 to 47, wherein the dsRNA is uracil.

54. The dsRNA according to claim 40, wherein the modified nucleotide is at least one of 5'-vinylphosphonate nucleotide, 5'-phosphate or phosphate mimetic, locked nucleic acid (LNA), 2'-MOE (methoxyethyl) nucleotide, and / or 2'-arabinofluoro (2'-araF) nucleotide.

55. The dsRNA according to claim 54, wherein the antisense strand comprises a phosphate mimetic at its 5' end, and optionally the phosphate mimetic is a 5'-E-vinyl-phosphonate or a 4'-O-phosphonate.

56. The dsRNA according to claim 40, wherein the modified nucleotide is at least one of nucleotides comprising a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a base-dropped nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, and / or a non-natural base.

57. The dsRNA according to claim 40, wherein the antisense strand and / or the sense strand comprises at least one nucleoside bond selected from the group consisting of phosphorothioate bond, phosphorodithioate bond, phosphotryester bond, alkylphosphonate bond, aminoalkylphosphotryester bond, alkylenephosphonate bond, phosphine bond, phosphoramidate bond, phosphormololate bond, phosphoropiperazidate bond, aminoalkylphosphoamidate bond, thiophosphoamidate bond, thionoalkylphosphonate bond, thionoalkylphosphotryester bond, thiophosphate bond, selenophosphate bond, and boranophosphate bond.

58. The dsRNA according to claim 57, wherein the antisense strand and / or the sense strand comprises at least one nucleotide modification linkage.

59. The dsRNA according to claim 57, wherein all nucleotide bonds in the antisense strand are modified bonds.

60. The dsRNA according to claim 57, wherein the antisense strand and / or the sense strand comprises at least one phosphorothioate (PS) linkage.

61. The dsRNA according to any one of claims 1 or 3 to 60, further comprising a ligand or targeting moiety.

62. The dsRNA according to claim 61, wherein the ligand or targeting portion is conjugated to the 5' end, 3' end, or both ends of the dsRNA.

63. The dsRNA according to claim 61, wherein the ligand or targeting portion is conjugated to the 3' end of the sense strand of the dsRNA.

64. The dsRNA according to claim 61, wherein the ligand or targeting portion is conjugated to the 5' end of the sense strand of the dsRNA.

65. The dsRNA according to any one of claim 61 or 64, wherein the ligand or targeting moiety is at least one N-acetyl-galactosamine (GalNAc).

66. The ligand or targeting portion is defined by the following formula (I): 【Transformation 5】 or represented by a pharmaceutically acceptable salt thereof, in the formula, A １ This is the binding site to the dsRNA, T １ and T ２ Each instance is independently selected from a 5-member heterocycline and alkylene. Each time X appears, it is selected from the group consisting of -OH and -SH. Each time L appears, it is a linker, L Ａ It either does not exist or is a linker. The dsRNA according to any one of claims 61 to 65, wherein n is an integer from 1 to 6.

67. T １ and T ２ Each instance independently contains a five-membered heterocycline and C, each having at least one ring oxygen. １－６ dsRNA according to any one of claims 61 to 66, selected from alkylenes.

68. The compound is represented by the following formula (I-A): 【Transformation 6】 The dsRNA according to any one of claims 61 to 67, or represented by a pharmaceutically acceptable salt thereof.

69. The aforementioned compound is given by the following formula (I-A-I): 【Transformation 7】 The dsRNA according to any one of claims 61 to 61, or represented by a pharmaceutically acceptable salt thereof.

70. The aforementioned compound is given by the following formula (I-A-II): 【Transformation 8】 The dsRNA according to any one of claims 61 to 56, or represented by a pharmaceutically acceptable salt thereof, wherein a and b are integers from 1 to 20 in each occurrence.

71. The dsRNA according to any one of claims 61 to 70, wherein the ligand or targeting portion is tri-GalNAc6.

72. The dsRNA according to any one of claims 61 to 65, wherein the ligand or targeting portion is L96.

73. A cell comprising the dsRNA described in any one of claims 1 to 72.

74. A vector encoding at least one unmodified strand of a dsRNA according to any one of claims 1 to 72, or optionally both strands.

75. A cell comprising the vector according to claim 74.

76. A pharmaceutical composition for inhibiting INHBE expression, comprising a dsRNA according to any one of claims 1 to 72 and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

77. A method for inhibiting intracellular INHBE expression, (a) Contacting the cells with the dsRNA described in any one of claims 1 to 72 or the pharmaceutical composition described in claim 76, (b) Maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the INHBE gene, thereby inhibiting the expression of the INHBE gene in the cells, the method optionally being in vivo.

78. The method according to claim 77, wherein the INHBE expression is inhibited by at least 30% compared to a control.

79. A method for treating a disorder mediated by or associated with INHBE, comprising administering to a subject in need of such treatment a therapeutically effective amount of dsRNA according to any one of claims 1 to 72 or the pharmaceutical composition according to claim 76.

80. The method according to claim 79, wherein the disorder is a cardiovascular disorder.

81. The method according to claim 79, wherein the disorder is a cardiovascular disease.

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