Inhibin subunit beta E (INHBE) modulator compositions and methods of use thereof

Abstract

The present invention relates to modulators, e.g., double-stranded RNA (dsRNA) agents, antisense polynucleotide agents, antibodies, guide RNAs that perform ADAR editing, or guide RNAs that perform CRISPR editing, that modulate, e.g., inhibit, the expression and / or activity of inhibin subunit beta E (INHBE). The present invention also relates to methods of using such modulators to inhibit the expression and / or activity of INHBE, and to methods of preventing and treating INHBE-related disorders, e.g., metabolic disorders, e.g., metabolic syndrome, in a subject.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 246,084, filed September 20, 2021, the entire contents of which are incorporated herein by reference.

[0002] This application is related to U.S. Provisional Patent Application No. 63 / 223,995, filed July 21, 2021, U.S. Provisional Patent Application No. 63 / 278,126, filed November 11, 2021, U.S. Provisional Patent Application No. 63 / 285,143, filed December 2, 2021, U.S. Provisional Patent Application No. 63 / 287,578, filed December 9, 2021, U.S. Provisional Patent Application No. 63 / 321,799, filed March 21, 2022, U.S. Provisional Patent Application No. 63 / 323,543, filed March 25, 2022, and PCT Publication No. PCT / US2022 / 037658, filed July 20, 2022. The entire contents of each of the foregoing applications are incorporated herein by reference. [Background technology]

[0003] Due to the successful eradication of many infectious diseases in most parts of the world, non-communicable diseases, specifically metabolic disorders, have become the major health hazards in the modern world. The increase in the consumption of high-calorie, low-fiber fast food and the decrease in physical activity due to mechanized transportation and sedentary lifestyles have led to the spread of metabolic disorders, such as metabolic syndrome, type 2 diabetes, hypertension, cardiovascular disease, stroke, and other physical disorders. In fact, the occurrence of subjects with metabolic disorders, such as metabolic syndrome, who have many health conditions that put them at high risk for heart disease, diabetes, stroke, and other diseases, has increased in recent years.

[0004] Current treatments for disorders of lipid metabolism include lifestyle changes, diet, exercise and treatment with drugs such as lipid lowering agents, e.g. statins, and other drugs.However, these treatments and treatments are often limited by compliance, are not always effective, cause side effects, and cause drug-drug interactions.Therefore, there is a need in the art for alternative treatments for subjects with metabolic disorders.

[0005] Inhibin subunit beta E (INHBE) is a member of the transforming growth factor-β (TGF-β) family. Primarily expressed in the liver, INHBE is a hepatokine that has been shown to positively correlate with insulin resistance and body mass index in humans. Quantitative real-time PCR analysis also showed increased INHBE gene expression in liver samples from insulin-resistant human subjects. Furthermore, inhbe gene expression was shown to be increased in the liver of the db / db mouse model, an art-accepted animal model of metabolic disorder, namely type 2 diabetes. Inhibition of inhbe expression in db / db mice was demonstrated to suppress weight gain, which was attributed to a decrease in obesity rather than lean mass. Summary of the Invention [Problem to be solved by the invention]

[0006] As discussed above, there is an unmet need for effective treatments for metabolic disorders such as metabolic syndrome, and related diseases, e.g., diabetes, hypertension, and cardiovascular disease, e.g., agents that can selectively and effectively modulate, i.e., inhibit, INHBE expression and / or activity. [Means for solving the problem]

[0007] The present invention provides, inter alia, modulators that modulate, ie, inhibit, the expression and / or activity of inhibin subunit beta E (INHBE) for treating INHBE-related disorders, such as metabolic disorders, such as metabolic syndrome.

[0008] In one embodiment, the present invention provides a modulator of inhibin subunit beta E (INHBE).The modulator can be an oligonucleotide that targets INHBE, such as a double-stranded ribonucleic acid (dsRNA) or an antisense polynucleotide agent; an antibody that specifically binds to INHBE, or an antigen-binding fragment thereof, such as a monoclonal anti-INHBE antibody, or an antigen-binding fragment thereof; a small molecule; a guide RNA that performs ADAR editing, such as a guide RNA that comprises a stem-loop structure that binds to ADAR enzyme; or a guide RNA that performs CRISPR editing.

[0009] In one embodiment, the antisense polynucleotide agent comprises 4 to 50 contiguous nucleotides, at least one of the contiguous nucleotides is a modified nucleotide, and the nucleotide sequence of the agent is 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 4, 6, 8, or 10.

[0010] In one embodiment, the equivalent region is any one of the target regions of SEQ ID NO:1 provided in Table 4.

[0011] In one embodiment, the antisense polynucleotide agent comprises at least 8 contiguous nucleotides that differ from any one of the nucleotide sequences listed in Table 3 by no more than 3 nucleotides.

[0012] In one embodiment, substantially all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.

[0013] In one embodiment, all of the nucleotides of the antisense polynucleotide agent are modified nucleotides.

[0014] In one embodiment, the antisense polynucleotide agent is 10 to 40 nucleotides in length.

[0015] In one embodiment, the antisense polynucleotide agent is 10-30 nucleotides in length.

[0016] In one embodiment, the antisense polynucleotide agent is 18-30 nucleotides in length.

[0017] In one embodiment, the antisense polynucleotide agent is 10-24 nucleotides in length.

[0018] In one embodiment, the antisense polynucleotide agent is 18-24 nucleotides in length.

[0019] In one embodiment, the antisense polynucleotide agent is 14-20 nucleotides in length.

[0020] In one embodiment, the antisense polynucleotide agent is 14 nucleotides in length.

[0021] In one embodiment, the antisense polynucleotide agent is 20 nucleotides in length.

[0022] In one embodiment, the modified nucleotide comprises a modified sugar moiety selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

[0023] In one embodiment, the bicyclic sugar moiety has a (-CH2-)n group forming a bridge between the 2' oxygen atom and the 4' carbon atom of the sugar ring, where n is 1 or 2 and R is H, CH3 or CH3OCH3.

[0024] In one embodiment, the modified nucleotide is 5-methylcytosine.

[0025] In one embodiment, the modified nucleotide comprises a modified internucleoside linkage.

[0026] In one embodiment, the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0027] In one embodiment, the modulator comprises a plurality of 2'-deoxynucleotides flanked on each side by at least one nucleotide having a modified sugar moiety.

[0028] In one embodiment, an antisense polynucleotide agent is a gapmer that includes a gap segment composed of linked 2'-deoxynucleotides positioned between the 5' and 3' wing segments.

[0029] In one embodiment, the modified sugar moiety is selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

[0030] In one embodiment, the 5'-wing segment is 1 to 6 nucleotides in length.

[0031] In one embodiment, the 3'-wing segment is 1 to 6 nucleotides in length.

[0032] In one embodiment, the gap segment is 5 to 14 nucleotides in length.

[0033] In one embodiment, the 5'-wing segment is 2 nucleotides in length.

[0034] In one embodiment, the 3'-wing segment is 2 nucleotides in length.

[0035] In one embodiment, the 5'-wing segment is 3 nucleotides in length.

[0036] In one embodiment, the 3'-wing segment is 3 nucleotides in length.

[0037] In one embodiment, the 5'-wing segment is 4 nucleotides in length.

[0038] In one embodiment, the 3'-wing segment is 4 nucleotides in length.

[0039] In one embodiment, the 5'-wing segment is 5 nucleotides in length.

[0040] In one embodiment, the 3'-wing segment is 5 nucleotides in length.

[0041] In one embodiment, the gap segment is 10 nucleotides in length.

[0042] In one embodiment, an antisense polynucleotide agent comprises a gap segment composed of linked deoxynucleotides; a 5'-wing segment composed of linked nucleotides; and a 3'-wing segment composed of linked nucleotides, wherein the gap segment is positioned between the 5'-wing segment and the 3'-wing segment, and each nucleotide of each wing segment comprises a modified sugar.

[0043] In one embodiment, the gap segment is 10 2'-deoxynucleotides in length and each wing segment is 5 nucleotides in length.

[0044] In one embodiment, the gap segment is 10 2'-deoxynucleotides in length and each wing segment is 4 nucleotides in length.

[0045] In one embodiment, the gap segment is 10 2'-deoxynucleotides in length and each wing segment is 3 nucleotides in length.

[0046] In one embodiment, the gap segment is 10 2'-deoxynucleotides in length and each wing segment is 2 nucleotides in length.

[0047] In one embodiment, the modified sugar moiety is selected from the group consisting of a 2'-O-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-O-alkyl modified sugar moiety, and a bicyclic sugar moiety.

[0048] In one embodiment, all of the nucleotides contain modified internucleoside linkages.

[0049] In one embodiment, the modulator further comprises a ligand.

[0050] In one embodiment, the modulator is conjugated to the ligand at the 3' end.

[0051] In one embodiment, the ligand is an N-acetylgalactosamine (GalNac) derivative.

[0052] In one embodiment, the ligand is

[0053] [ka] It is.

[0054] The present invention also provides cells containing any of the modulators of the present invention and pharmaceutical compositions comprising any of the modulators of the present invention.

[0055] The pharmaceutical compositions of the invention may comprise the modulator in a non-buffered solution, such as saline or water, and the pharmaceutical compositions of the invention may comprise the modulator in a buffer, such as a buffer comprising acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof; or phosphate buffered saline (PBS). In some embodiments, the pharmaceutical composition comprises the modulator and a lipid formulation, for example, the lipid formulation comprises LNP or the lipid formulation comprises MC3.

[0056] In one aspect, the present invention provides a method of inhibiting expression and / or activity of inhibin subunit beta E (INHBE) in a cell, the method comprising contacting the cell with either a modulator of the present invention or a pharmaceutical composition of the present invention, thereby inhibiting expression and / or activity of the INHBE gene in the cell.

[0057] In one embodiment, the cell is, for example, a cell within a human subject, for example, a subject having a metabolic disorder, such as diabetes, or a cardiovascular disease, such as hypertension.

[0058] In certain embodiments, INHBE expression and / or activity is inhibited by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In one embodiment, inhibiting INHBE expression and / or activity reduces INHBE protein levels in the serum of the subject by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0059] In one aspect, the present invention provides a method of treating a subject having a disorder that would benefit from reduced inhibin subunit beta E (INHBE) expression and / or activity. The method comprises administering to the subject a therapeutically effective amount of either a modulator of the present invention, or a pharmaceutical composition of the present invention, thereby treating the subject having a disorder that would benefit from reduced INHBE expression.

[0060] In another aspect, the present invention provides a method of preventing at least one symptom in a subject having a disorder that would benefit from reduced inhibin subunit beta E (INHBE) expression and / or activity. The method comprises administering to the subject a prophylactically effective amount of either a modulator of the present invention, or a pharmaceutical composition of the present invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from reduced INHBE expression.

[0061] In one embodiment, administration of a therapeutically or prophylactically effective amount reduces waist-to-hip ratio in line with body mass index in a subject.

[0062] In certain embodiments, the disorder is a metabolic disorder, such as metabolic syndrome, carbohydrate disorders, such as type II diabetes, prediabetes, lipid disorders, such as hyperlipidemia, hypertension, cardiovascular disease, weight disorders.

[0063] In some embodiments, the INHBE-related disorder is metabolic syndrome.

[0064] In some embodiments, the INHBE-related disorder is a cardiovascular disease.

[0065] In some embodiments, the INHBE-related disorder is hypertension.

[0066] In certain embodiments, administration of a modulator to a subject causes a reduction in INHBE protein accumulation in the subject.

[0067] In a further embodiment, the present invention also provides a method of inhibiting expression and / or activity of INHBE in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the modulators presented herein, thereby inhibiting expression and / or activity of INHBE in the subject.

[0068] In one embodiment, the subject is a human.

[0069] In one embodiment, the modulator is administered to a subject at a dose of from about 0.01 mg / kg to about 50 mg / kg.

[0070] In one embodiment, the modulator is administered subcutaneously to the subject.

[0071] In one embodiment, the method of the invention further comprises determining the level of INHBE in a sample(s) from the subject.

[0072] In one embodiment, the level of INHBE in the subject sample(s) is the INHBE protein level in blood sample(s) or serum sample(s) or liver tissue sample(s).

[0073] In certain embodiments, the methods of the invention further comprise administering to the subject an additional therapeutic agent.

[0074] In certain embodiments, the additional therapeutic agent is selected from the group consisting of insulin, glucagon-like peptide 1 agonists, sulfonylureas, seglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and any combination of the above.

[0075] The invention also provides kits comprising either a modulator of the invention, or a pharmaceutical composition of the invention, and optionally, instructions for use. In one embodiment, the invention provides a kit for carrying out a method of inhibiting INHBE expression and / or activity in a cell by contacting the cell with a modulator of the invention in an amount effective to inhibit INHBE expression and / or activity in the cell. The kit comprises the modulator and instructions for use, and optionally, a means for administering the modulator to a subject. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] The present invention provides compositions comprising modulators, i.e. inhibitors, of the Inhibin subunit beta E (INHBE) gene for treating Inhibin subunit beta E (INHBE) related disorders, e.g., metabolic disorders, e.g., metabolic syndrome, carbohydrate metabolism disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertension, cardiovascular disease, body weight disorders.

[0077] The "Description of the Invention" below discloses how to make and use compositions containing modulators to inhibit INHBE expression and / or activity, as well as compositions, uses, and methods for treating subjects who would benefit from inhibition and / or reduction of INHBE expression and / or activity, e.g., subjects susceptible to or diagnosed with an INHBE-associated disorder.

[0078] I. Definition So that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values ​​for a parameter is recited, the values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0079] As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0080] As used herein, the term "including" is used to mean, and is used interchangeably with, the phrase "including but not limited to."

[0081] As used herein, the term "or" is used to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. For example, "sense or antisense strand" is understood to mean "sense or antisense strand, or sense and antisense strand."

[0082] The term "about" is used herein to mean within typical tolerances in the art. For example, "about" may be understood to be about 2 standard deviations from the mean. In certain embodiments, "about" means ±10%. In certain embodiments, "about" means ±5%. When "about" is present before a series of numbers or ranges, it is understood that "about" may modify each of the series of numbers or ranges of numbers.

[0083] The term "at least," "more than," or "or more than" before a number or series of numbers is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may be logically included as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 nucleotides of a 21-nucleotide nucleic acid molecule" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the series of numbers or range of numbers.

[0084] As used herein, "no more than" or "or less" is understood to refer to the value adjacent to this phrase and to values ​​or integers logically less than it, up to zero, as is logical from the context. For example, a duplex with an overhang of "no more than 2 nucleotides" has an overhang of 2, 1, or 0 nucleotides. When "no more than" is present after a series of numbers or ranges, it is understood that the "no more than" can modify each of the series of numbers or ranges of numbers. As used herein, ranges include both the upper and lower limits.

[0085] As used herein, a detection method may include the determination that the amount of analyte present is below the detection level of the method.

[0086] In the event of a discrepancy between the nucleotide sequence of a designated target site and the nucleotide sequence of the sense or antisense strand, the designated sequence takes precedence.

[0087] In the event of a discrepancy between the sequence and its site indicated on the transcript or other sequences, the nucleotide sequence listed herein takes precedence.

[0088] As used herein, a "modulator" is a molecule that reduces or increases the expression and / or activity of INHBE.

[0089] As used herein, "inhibin subunit beta E" is used interchangeably with the term "INHBE" and refers to a growth factor belonging to the transforming growth factor-β (TGF-β) family. INHBE mRNA is mainly expressed in the liver (Fang J. et al. Biochemical & Biophysical Res. Comm. 1997; 231(3):655-61), and INHBE is involved in the control of liver cell proliferation and differentiation (Chabicovsky M. et al. Endocrinology. 2003; 144(8):3497-504). INHBE is also known as inhibin beta E chain, activin beta E, inhibin beta E subunit, inhibin beta E, and MGC4638.

[0090] The sequence of the human INHBE mRNA transcript can be found, for example, in GenBank Accession No. GI:1877089956 (NM_031479.5; SEQ ID NO:1; reverse complement, SEQ ID NO:2). The sequence of the mouse INHBE mRNA can be found, for example, in GenBank Accession No. GI:1061899809 (NM_008382.3; SEQ ID NO:3; reverse complement, SEQ ID NO:4). The sequence of the rat INHBE mRNA can be found, for example, in GenBank Accession No. GI:148747589 (NM_031815.2; SEQ ID NO:5; reverse complement, SEQ ID NO:6). The predicted sequence of rhesus monkey (Macaca mulatta) INHBE mRNA can be found, for example, in GenBank Accession No. GI:1622845604 (XM_001115958.3; SEQ ID NO: 7; reverse complement, SEQ ID NO: 8).

[0091] Further examples of INHBE mRNA sequences are readily available via public databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0092] Further information about INHBE can be found, for example, at www.ncbi.nlm.nih.gov / gene / ?term=INHBE.

[0093] Each of the foregoing GenBank Accession Numbers and Gene Database Numbers is hereby incorporated by reference in its entirety as of the filing date of this application.

[0094] As used herein, the term INHBE also refers to the mutation of INHBE gene, including the variants provided in SNP databases.Many sequence variations in INHBE gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?term=INHBE, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0095] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed upon transcription of the INHBE gene, including mRNA that is the product of RNA processing of the primary transcript.

[0096] In one embodiment, the target portion of the sequence will be at least sufficiently long to serve as a substrate for iRNA-directed cleavage at or near this portion of the nucleotide sequence of the mRNA molecule formed upon transcription of the INHBE gene. In another embodiment, the target sequence is a nucleic acid molecule to which an antisense polynucleotide agent of the invention specifically hybridizes.

[0097] The target sequence can be about 19-36 nucleotides in length, e.g., about 19-30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, and may be 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated as part of this disclosure.

[0098] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide that comprises a strand of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0099] "G", "C", "A", "T" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, or surrogate replacement moieties (see, for example, Table 1), which are described in more detail below. Those skilled in the art will be well aware that guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide that contains a nucleotide carrying such a replacement moiety. For example, and without limitation, a nucleotide that contains inosine as its base can base pair with a nucleotide that contains adenine, cytosine or uracil. Thus, a nucleotide that contains uracil, guanine or adenine can be replaced, for example, by a nucleotide that contains inosine within the nucleotide sequence of an oligonucleotide featured in the present invention. In another example, adenine and cytosine at any position within an oligonucleotide can be replaced by guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

[0100] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent", as used herein interchangeably, refer to an agent that contains RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway, as this term is defined herein. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA modulates, e.g., inhibits, the expression of INHBE gene in cells, e.g., liver cells, within a subject, such as a mammalian subject.

[0101] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a target mRNA sequence of INHBE, to direct the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNAs by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes dsRNA into 19-23 base pair short interfering RNAs with characteristic 2-base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one aspect, the present invention relates to single-stranded RNA (siRNA) that is generated in cells and promotes the formation of a RISC complex that performs the silencing of a target gene, i.e., the INHBE gene. Thus, in this specification, the term "siRNA" is also used to refer to the iRNA described above.

[0102] In certain embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as single-stranded siRNAs, chemically modified by the methods described herein or Lima et al., (2012) Cell 150:883-894.

[0103] In certain embodiments, the "iRNA" for use in the compositions, uses, and methods of the present invention is double-stranded RNA, and is referred to herein as a "double-stranded RNA agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure that includes two antiparallel, substantially complementary nucleic acid strands, and is referred to as having a "sense" and "antisense" orientation relative to the target RNA, i.e., the INHBE gene. In some embodiments of the present invention, the double-stranded RNA (dsRNA) induces the degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0104] Generally, the majority of the nucleotides in each strand of the oligonucleotide, e.g., dsRNA molecule of the present invention are ribonucleotides, but each strand or both strands may also contain one or more non-ribonucleotides, e.g., deoxyribonucleotides or modified nucleotides, as described in detail herein.In addition, the "iRNA" or "antisense polynucleotide agent" used herein may contain ribonucleotides with chemical modifications; the iRNA or antisense polynucleotide agent may contain substantial modifications in multiple nucleotides.The term "modified nucleotide" used herein refers to a nucleotide that has independently modified sugar moieties, modified internucleoside linkages, or modified nucleobases, or any combination thereof.Thus, the term modified nucleotide includes, for example, the substitution, addition, or removal of functional groups or atoms from internucleoside linkages, sugar moieties, or nucleobases.Modifications suitable for use in the agent of the present invention include all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA type molecules are encompassed by "iRNA" or "RNAi agent" or "antisense polynucleotide agent" for purposes of the specification and claims.

[0105] In certain embodiments of the present disclosure, the incorporation of deoxynucleotides can be considered to constitute modified nucleotides when present in an RNAi agent or an antisense polynucleotide agent.

[0106] The duplex region of the RNAi agent can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can be about 19-36 base pairs in length, e.g., about 19-30 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, about 19-36 base pairs in length, e.g. ... The length of the duplex region may range from 0, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, etc. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the ranges and lengths recited above are also contemplated as part of the present disclosure.

[0107] The two strands forming the duplex structure of the RNAi molecule may be different parts of one larger RNA molecule or may be separate RNA molecules. When the two strands are part of one larger molecule and are connected by an uninterrupted nucleotide chain that thus forms a duplex structure between the 3' end of one strand and the 5' end of the other strand, the connecting RNA strand is referred to as a "hairpin loop". A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23, or more unpaired nucleotides. In some embodiments, a hairpin loop may be 10 nucleotides or less. In some embodiments, a hairpin loop may be 8 unpaired nucleotides or less. In some embodiments, a hairpin loop may be 4-10 unpaired nucleotides. In some embodiments, a hairpin loop may be 4-8 nucleotides.

[0108] When two substantially complementary strands of dsRNA are contained in separate RNA molecules, these molecules do not have to be covalently connected, but may be. When two strands are covalently connected by means other than an uninterrupted chain of nucleotides that forms a duplex structure between the 3' end of one strand and the 5' end of the other strand, the connecting structure is called a "linker". The RNA strands may have the same number of nucleotides or may have different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest dsRNA strand minus any overhangs present in the duplex. In addition to the duplex structure, the RNAi may include one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides, for example, a 3' overhang of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two nucleotides, for example, a 5' overhang of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0109] In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which contains 19-23 nucleotides, that interacts with a target RNA sequence, eg, the INHBE gene, to direct cleavage of the target RNA.

[0110] In some embodiments, an iRNA of the disclosure is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a target mRNA sequence of INHBE, to direct cleavage of the target RNA.

[0111] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that overhangs from the double-stranded structure of a double-stranded iRNA. For example, a nucleotide overhang exists when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides. A nucleotide overhang can include or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang(s) can be on the sense strand, on the antisense strand, or on any combination thereof. Furthermore, a nucleotide overhang(s) can be present on the 5' end, on the 3' end, or on both ends of the antisense or sense strand of a dsRNA.

[0112] In one embodiment, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' or 5' end. In one embodiment, the sense strand of the dsRNA has an overhang of 1-10 nucleotides, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' or 5' end. In another embodiment, one or more of the nucleotides in the overhang are replaced by a thiophosphate nucleoside.

[0113] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, e.g., 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' or 5' end. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3' or 5' end. In another embodiment, one or more of the nucleotides in the overhang are replaced by a thiophosphate nucleoside.

[0114] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at the 3'-end or 5'-end. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include a length that extends beyond a length of 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides. In certain embodiments, the overhang is present on the sense strand of the duplex. In certain embodiments, the overhang is present on the 3'-end of the sense strand of the duplex. In certain embodiments, the overhang is present on the 5'-end of the sense strand of the duplex. In certain embodiments, the overhang is present on the antisense strand of the duplex. In certain embodiments, the overhang is present on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present on the 5'-end of the antisense strand of the duplex.In certain embodiments, one or more of the nucleotides in the extended overhang are replaced by thiophosphate nucleosides.In certain embodiments, the overhang comprises a self-complementary portion, so that the overhang can form a hairpin structure that is stable under physiological conditions.

[0115] "Blunt" or "blunt end" means that there is no unpaired nucleotide at this end of the double-stranded RNA agent, i.e., there is no nucleotide overhang.A "blunt-ended" double-stranded RNA agent is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule.The RNAi agent of the present invention includes an RNAi agent without a nucleotide overhang at one end (i.e., an agent with one overhang and one blunt end), or an RNAi agent without a nucleotide overhang at either end.Such a molecule will most likely be double-stranded throughout its entire length.

[0116] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA strand, that includes a region that is substantially complementary to a target sequence, e.g., INHBE mRNA.

[0117] As used herein, the term "complementarity region" refers to a region on the antisense strand of a dsRNA agent or a region of an antisense polynucleotide agent that is substantially complementary to a sequence, e.g., a target sequence, e.g., an INHBE nucleotide sequence as defined herein. If the complementary region is not completely complementary to the target sequence, the mismatch may be in an internal region of the molecule or in a terminal region. In general, the most tolerable mismatch is in the terminal region, e.g., within 5, 4, 3 nucleotides from the 5' or 3' end of the iRNA. In some embodiments, the double-stranded RNA agent or antisense polynucleotide agent of the invention contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent or antisense polynucleotide agent of the invention contains 4 or fewer mismatches with the target mRNA, e.g., the antisense strand contains 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the invention contains no more than four mismatches with the sense strand, e.g., the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the invention contains no more than four mismatches with the antisense strand, e.g., the sense strand contains four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is within, e.g., 5, 4, 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is within, e.g., the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(es) is not within the seed region.

[0118] Thus, the RNAi agent or antisense polynucleotide agent described herein may contain one or more mismatches with the target sequence. In one embodiment, the RNAi agent or antisense polynucleotide agent described herein contains three or less mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agent or antisense polynucleotide agent described herein contains two or less mismatches. In one embodiment, the RNAi agent or antisense polynucleotide agent described herein contains one or less mismatches. In one embodiment, the RNAi agent or antisense polynucleotide agent described herein contains zero mismatches. In certain embodiments, when the antisense strand of the RNAi agent or antisense polynucleotide agent contains a mismatch with the target sequence, the mismatch may be constrained to within the last 5 nucleotides from the 5' or 3' end of the complementary region. For example, in such an embodiment, for a 23 nucleotide RNAi agent, the strand that is complementary to a region of the INHBE gene generally does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an RNAi agent or antisense polynucleotide agent containing a mismatch with a target sequence is effective in inhibiting the expression of the INHBE gene. The effectiveness of an RNAi agent or antisense polynucleotide agent with a mismatch in inhibiting the expression of the INHBE gene is important, especially when a particular complementary region in the INHBE gene is known to have polymorphic sequence variation in the population.

[0119] As used herein, the term "sense strand" or "passenger strand," as that term is defined herein, refers to an iRNA strand that includes a region that is substantially complementary to a region of the antisense strand.

[0120] As used herein, "substantially all of the nucleotides are modified" means that the majority, but not all, are modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0121] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site where nucleotides 10 and 11 of the antisense strand are bound, and the cleavage region comprises nucleotides 11, 12, and 13.

[0122] As used herein, and unless otherwise indicated, the term "complementarity", when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions, as understood by those skilled in the art. Such conditions may be stringent conditions, which may include, for example, 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50°C or 70°C for 12-16 hours, followed by washing [see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press]. Other conditions, such as physiologically relevant conditions that may be encountered inside an organism, may also be applied. Those skilled in the art will be able to determine the most appropriate set of conditions to test for the complementarity of two sequences according to the ultimate application of the nucleotides to be hybridized.

[0123] The complementary sequence described herein includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both of the nucleotide sequences. In this specification, such sequences can be referred to as "fully complementary" with respect to each other. However, when a first sequence is referred to as "substantially complementary" with respect to a second sequence, the two sequences can be fully complementary or can form one or more mismatched base pairs, generally not more than 5, 4, 3, or 2, upon hybridization for a duplex of up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their final application, e.g., the inhibition of gene expression in vitro or in vivo. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered as mismatches in terms of determining complementarity. For example, a dsRNA containing a 21-nucleotide sequence, including one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, may also be referred to as "fully complementary" for purposes described herein.

[0124] "Complementary" sequences as used herein may also include or be formed entirely of non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0125] As used herein, the terms "complementarity," "fully complementary," and "substantially complementary" may be used in reference to base matching between two oligonucleotides or polynucleotides, such as a sense strand and an antisense strand of a dsRNA, or an antisense strand of a double-stranded RNA agent and a target sequence, as understood from the context of their use.

[0126] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding an INHBE gene). For example, a polynucleotide is complementary to at least a portion of an INHBE mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding the INHBE gene.

[0127] Thus, in some embodiments, the antisense polynucleotide disclosed herein is fully complementary to the target INHBE sequence. In other embodiments, the antisense polynucleotide disclosed herein is substantially complementary to the target INHBE sequence, and comprises a contiguous nucleotide sequence that is at least 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary to the equivalent region of any one of the nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, or 9, or a fragment of any one of SEQ ID NO: 1, 3, 5, 7, or 9, over its entire length.

[0128] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target INHBE sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-5, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-5.

[0129] In one embodiment, an RNAi agent of the disclosure comprises an antisense polynucleotide and a sense strand that is substantially complementary, where the antisense polynucleotide is the same as the targeted INHBE sequence, where the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary to the equivalent region of the nucleotide sequence of SEQ ID NO:2, 4, 6, 8, or 10, or a fragment of any one of SEQ ID NO:2, 4, 6, 8, or 10, over its entire length.

[0130] In some embodiments, an iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide, where the antisense polynucleotide is complementary to a target INHBE sequence, where the sense strand polynucleotide comprises a contiguous nucleotide sequence over its entire length that is at least about 80% complementary, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary to any one of the antisense strand nucleotide sequences in any one of Tables 2-3, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-3.

[0131] Generally, "iRNA" comprises ribonucleotides with chemical modifications. Such modifications may include all kinds of modifications disclosed herein or known in the art. Any such modifications used in dsRNA molecules are encompassed by "iRNA" for the purposes of this specification and claims.

[0132] In certain embodiments of the present disclosure, incorporation of deoxynucleotides when present in an RNAi agent is considered to constitute modified nucleotides.

[0133] The terms "polynucleotide agent", "antisense polynucleotide agent", "antisense compound" and "agent" are used interchangeably herein, as the term is defined herein, refer to an agent that contains RNA and includes a single-stranded oligonucleotide that targets a nucleic acid molecule that codes for INHBE (e.g., the mRNA that codes for INHBE, as provided in any one of SEQ ID NOs: 1, 3, 5, 7, or 9). Antisense polynucleotide agents specifically bind to target nucleic acid molecules via hydrogen bonds (e.g., Watson-Crick, Hoogstein, or reverse Hoogstein hydrogen bonds) and interfere with the normal function of the target nucleic acid (e.g., by an antisense mechanism of action). Interference with or modulation of the function of a target nucleic acid by a polynucleotide agent of the present invention is referred to as "antisense inhibition".

[0134] The function of the target nucleic acid molecule that is interfered with can include, for example, functions such as translocation of RNA to a protein translation site, translation of protein from RNA, splicing of RNA to generate one or more mRNA species, and catalytic activities that can be engaged in and promoted by RNA.

[0135] In some embodiments, antisense inhibition refers to "inhibiting expression" of target nucleic acid level or target protein level in a cell, such as a mammalian subject, in a cell, in a subject, in the presence of an antisense polynucleotide agent that is complementary to the target nucleic acid, compared to the target nucleic acid level or target protein level in the absence of the antisense polynucleotide agent.For example, the antisense polynucleotide agent of the present invention can inhibit translation in a stoichiometric manner by physically blocking base pairing with mRNA and translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355.

[0136] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. The term antibody as used herein refers to a molecule that contains at least the complementarity determining region (CDR)1, CDR2, and CDR3 of a single domain antibody (sdAb), which can bind to an antigen. The term antibody also refers to a molecule that contains at least the CDR1, CDR2, and CDR3 of a heavy chain, and the CDR1, CDR2, and CDR3 of a light chain, which can bind to an antigen. The term antibody also includes fragments that can bind to an antigen, such as scFv, single chain Fv (scFv), Fab, Fab', and (Fab')2. The term antibody also includes chimeric antibodies, humanized antibodies, and antibodies of various species, such as mouse, human, cynomolgus monkey, llama, camel, etc. The term also includes multivalent antibodies, such as bivalent or tetravalent antibodies. Multivalent antibodies include, for example, a single polypeptide chain comprising multiple antigen-binding (CDR-containing) domains, as well as two or more polypeptide chains, each containing one or more antigen-binding domains, and associated with each other, for example, by a hinge region which can form disulfide bond(s) or any other covalent or non-covalent interaction.

[0137] The term "heavy chain variable region" as used herein refers to a region comprising heavy chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the heavy chain variable region also comprises at least a portion of FR1 and / or a portion of FR4. In some embodiments, heavy chain CDR1 corresponds to Kabat residues 26-35; heavy chain CDR2 corresponds to Kabat residues 50-65; and heavy chain CDR3 corresponds to Kabat residues 95-102. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.); and FIG. 1. In some embodiments, heavy chain CDR1 corresponds to Kabat residues 31-35; heavy chain CDR2 corresponds to Kabat residues 50-65; and heavy chain CDR3 corresponds to Kabat residues 95-102. See ibid.

[0138] The term "heavy chain constant region" as used herein refers to a region that includes at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody that includes a gamma constant region is an IgG antibody, an antibody that includes a delta constant region is an IgD antibody, and an antibody that includes an alpha constant region is an IgA antibody. Furthermore, an antibody that includes a mu constant region is an IgM antibody, and an antibody that includes an epsilon constant region is an IgE antibody. A particular isotype can be further divided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a gamma 1 constant region), IgG2 (containing a gamma 2 constant region), IgG3 (containing a gamma 3 constant region), and IgG4 (containing a gamma 4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an alpha 1 constant region) and IgA2 (containing an alpha 2 constant region) antibodies; IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0139] As used herein, the term "heavy chain" (abbreviated HC) refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. As used herein, the term "full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence.

[0140] As used herein, the term "light chain variable region" refers to a region comprising light chain CDR1, framework (FR) 2, CDR2, FR3, and CDR3. In some embodiments, the light chain variable region also comprises FR1 and / or FR4. In some embodiments, light chain CDR1 corresponds to Kabat residues 24-34; light chain CDR2 corresponds to Kabat residues 50-56; and light chain CDR3 corresponds to Kabat residues 89-97. See, e.g., Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.).

[0141] As used herein, the term "light chain constant region" refers to the region that contains the light chain constant domain, CL. Non-limiting exemplary light chain constant regions include λ and κ.

[0142] As used herein, the term "light chain" (abbreviated LC) refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. As used herein, the term "full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.

[0143] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds INHBE is substantially free of antibodies that specifically bind antigens other than INHBE). However, an isolated antibody that specifically binds INHBE may have cross-reactivity with other antigens, such as INHBE molecules from other species. Furthermore, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0144] As used herein, a "chimeric antibody" refers to an antibody that comprises at least one variable region from a first species (e.g., mouse, rat, cynomolgus monkey, etc.) and at least one constant region from a second species (e.g., human, cynomolgus monkey, etc.). In some embodiments, a chimeric antibody comprises at least one mouse variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one cynomolgus monkey variable region and at least one human constant region. In some embodiments, a chimeric antibody comprises at least one rat variable region and at least one mouse constant region. In some embodiments, all of the variable regions of a chimeric antibody are from a first species and all of the constant regions of a chimeric antibody are from a second species.

[0145] As used herein, a "humanized antibody" refers to an antibody in which at least one amino acid in the framework region of a non-human variable region is replaced with the corresponding amino acid from a human variable region. In some embodiments, the humanized antibody comprises at least one human constant region or a fragment thereof. In some embodiments, the humanized antibody is an sdAb, Fab, scFv, (Fab')2, etc. The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3 and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and the particular constant domain may be selected to optimize desired effector functions using techniques well known in the art.

[0146] As used herein, "human antibody" refers to antibodies produced in humans, antibodies produced in non-human animals that contain human immunoglobulin genes, such as the XenoMouse®, and antibodies selected using in vitro methods, such as phage display, whose antibody repertoires are based on human immunoglobulin sequences.

[0147] The terms "antibody that specifically binds to INHBE, or an antigen-binding fragment thereof" or "anti-INHBE antibody or an antigen-binding fragment thereof" are used interchangeably herein and refer to an antibody, or an antigen-binding fragment thereof, that specifically binds to INHBE, e.g., human INHBE. An antibody that "binds" to an antigen of interest, i.e., INHBE, is one that can bind to the antigen with sufficient affinity such that the antibody is useful for targeting cells expressing the antigen. In certain embodiments, the antibody specifically binds to human INHBE. Unless otherwise indicated, the term "anti-INHBE antibody" is meant to refer to an antibody that binds to wild-type INHBE, mutants, or isoforms of INHBE.

[0148] In one embodiment, the phrase "specifically binds to INHBE" or "specifically bound to INHBE" as used herein means about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 75 nM or less, about 25 nM or less, about 21 nM or less, about 12 nM or less, about 11 nM or less. , about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.3 nM or less, about 0.1 nM or less, about 0.01 nM or less, or about 0.001 nM or less of the dissociation constant (K D In another embodiment, the phrase "specifically binds to INHBE" or "specifically bound to INHBE" as used herein refers to the ability of an anti-INHBE antibody to interact with INHBE with a dissociation constant (K) of between about 1 pM (0.001 nM) and 2000 nM, between about 500 pM (0.5 nM) and 1000 nM, between about 500 pM (0.5 nM) and 500 nM, between about 1 nM and 200 nM, between about 1 nM and 100 nM, between about 1 nM and 50 nM, between about 1 nM and 20 nM, or between about 1 nM and 5 nM. D ) refers to the ability of an anti-INHBE antibody to interact with INHBE. D is determined by surface plasmon resonance or by any other method known in the art.

[0149] The terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. As recognized in the art, these terms refer to a system of numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31-35 in CDR1, amino acid positions 50-65 in CDR2, and amino acid positions 95-102 in CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0150] As used herein, the term "CDR" refers to the complementarity determining region in antibody variable sequence. There are three CDRs in each of the variable regions of heavy chain (HC) and light chain (LC), and for each of the variable regions, they are named CDR1, CDR2 and CDR3 (or specifically, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3). As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that can bind to antigen. The exact boundaries of these CDRs are defined separately according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) have described the Kabat CDRs as We found that certain subpopulations within CDRs have great diversity at the level of amino acid sequence, but adopt almost identical peptide backbone structures. These subpopulations are named L1, L2 and L3 or H1, H2 and H3, with "L" and "H" designating the light chain region and the heavy chain region, respectively. These regions are called Chothia CDRs, and have boundaries that overlap with Kabat CDRs. Other boundaries that define the CDRs that overlap with Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).Still other CDR boundary definitions may not strictly follow one of the above systems, but may be shortened or lengthened in light of predictions or experimental findings that, despite overlap with the Kabat CDRs, certain residues or groups of residues or even entire CDRs do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs.

[0151] As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. The exact definition of the CDR sequence can be determined by different systems, so the meaning of the framework sequence is interpreted differently accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain, and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions of the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) of each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR4. Without specifying a particular subregion as FR1, FR2, FR3, or FR4, the framework region represents the combined FRs in the variable region of a single naturally occurring immunoglobulin chain, as mentioned elsewhere. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that form a framework region.

[0152] The framework and CDR regions of a humanized antibody need not correspond exactly to the parent sequence, e.g., donor antibody CDR, or the consensus framework may be mutated by substitution, insertion and / or deletion of at least one amino acid residue, such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in a preferred embodiment, such mutations will not be extensive. Usually, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to those of the parent FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region of a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0153] The term "epitope" refers to the region of an antigen bound by an antibody or antibody fragment. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments may have specific three dimensional structural characteristics, and / or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0154] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biomolecular specific interactions by detecting changes in protein concentration in a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor ab, Uppsala, Sweden and Piscataway, NJ).For further explanation, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0155] As used herein, "K on " or "K a The term "on rate constant" is intended to refer to the on rate constant for the association of an antibody to an antigen to form an antibody / antigen complex.

[0156] As used herein, "K off " or "K d The term "off rate constant" is intended to refer to the off rate constant for dissociation of an antibody from the antibody / antigen complex.

[0157] As used herein, "K D The term " is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. D is K a / K dIn one embodiment, an antibody of the invention has a K of about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 200 nM or less, about 100 nM or less, about 75 nM or less, about 25 nM or less, about 21 nM or less, about 12 nM or less, about 11 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.3 nM or less, about 0.1 nM or less, about 0.01 nM or less, or about 0.001 nM or less. D has.

[0158] As used herein, the phrase "contacting a cell with a modulator", such as an antisense polynucleotide agent, includes contacting a cell by any possible means. Contacting a cell with a modulator includes contacting a cell with a modulator in vitro or contacting a cell with a modulator in vivo. Contact can be direct or indirect. Thus, for example, the modulator can be physically contacted with a cell by the person performing the method, or alternatively, the modulator can be placed in a situation that allows it to contact the cell or that allows it to contact the cell thereafter.

[0159] The contact of cells in vitro can be, for example, by incubating the cells with the modulator. The contact of cells in vivo can be, for example, by injecting the modulator into or near the tissue where the cells are located, or by injecting the modulator into another area, for example, into the bloodstream or subcutaneous cavity, so that the modulator then reaches the tissue where the cells to be contacted are located. For example, the modulator, for example, iRNA, can contain or be coupled with a ligand, for example, GalNAc, that directs the iRNA to the desired site, for example, the liver. The combination of the contacting method in vitro and the contacting method in vivo is also possible. For example, the cell can also be contacted with the modulator in vitro and then transplanted into a subject.

[0160] In certain embodiments, contacting a cell with a modulator includes "introducing a modulator into a cell" or "delivering a modulator into a cell" by facilitating or resulting in uptake or absorption into the cell. Absorption or uptake of iRNA may occur via spontaneous diffusion or active intracellular processes, or may occur with ancillary agents or devices. Introduction of a modulator into a cell may be in vitro or in vivo. For example, for in vivo introduction, the modulator may be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further techniques are described herein below or known in the art.

[0161] The term "lipid nanoparticle" or "LNP" refers to a vesicle that includes a lipid layer that encapsulates a pharmacoactive molecule, such as a nucleic acid molecule, e.g., an iRNA, or a plasmid into which an iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0162] As used herein, a "subject" is an animal, such as a mammal, including a primate (human, non-human primate, such as monkey and chimpanzee), non-primate (e.g., cow, pig, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, or mouse), or bird, that expresses a target gene endogenously or heterologously. In certain embodiments, the subject is a human, such as a human being treated for or evaluated for a disease or disorder that would benefit from reduced INHBE expression and / or activity, as described herein; a human being at risk for a disease or disorder that would benefit from reduced INHBE expression and / or activity; a human having a disease or disorder that would benefit from reduced INHBE expression and / or activity; or a human being treated for a disease or disorder that would benefit from reduced INHBE expression and / or activity. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

[0163] As used herein, the term "treating" or "treatment" refers to a beneficial or desired outcome, such as a reduction in at least one sign or symptom of an INHBE-associated disorder in a subject. Treatment also includes alleviating one or more signs or symptoms associated with undesired INHBE expression and / or activity; reducing the degree of undesired INHBE activation or stabilization; ameliorating or alleviating undesired INHBE activation or stabilization. "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0164] The term "reduction" in the context of the level or symptoms of an INHBE or disease marker in a subject refers to a statistically significant reduction in such level. The reduction can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more reduction. In certain embodiments, the reduction is at least 20%. In certain embodiments, the reduction is at least a 50% reduction in a disease marker, e.g., protein level or gene expression level. In the context of INHBE levels in a subject, "reduction" is a reduction to a level that is accepted as being within the normal range for individuals without such a disorder. In certain embodiments, "reduction" is a reduction in the difference between the level or symptoms of a marker in a subject suffering from a disease and a level that is accepted as being within the normal range for an individual. The term "reduction" may also be used in connection with normalizing a symptom of a disease or condition, i.e., reducing the difference between the level in a subject suffering from an INHBE-associated disorder and the level in a normal subject not suffering from an INHBE-associated disorder. As used herein, when a disease is associated with an elevated value for a symptom, "normal" is considered to be the upper limit of normal. When a disease is associated with a decreased value for a symptom, "normal" is considered to be the lower limit of normal.

[0165] As used herein, "prevention" or "preventing" when used in reference to a disease, disorder, or condition that can be treated or alleviated by reducing the expression and / or activity of INHBE refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, such as symptoms of an INHBE-associated disorder, such as a metabolic disorder, such as diabetes. A failure to develop a disease, disorder, or condition, or a reduction in the development of symptoms associated with such a disease, disorder, or condition (e.g., at least about 10% on a clinically acceptable scale for the disease or disorder), or a delayed presentation of symptoms (e.g., by days, weeks, months, or years) is considered effective prevention.

[0166] As used herein, the term "inhibin subunit beta E-related disorder" or "INHBE-related disorder" is a disease or disorder caused by or associated with expression of the INHBE gene or INHBE protein production and / or activity. The term "INHBE-related disorder" includes diseases, disorders, or conditions that benefit from reduced expression, replication, or protein activity of the INHBE gene. In some embodiments, the INHBE-related disorder is a metabolic disorder, e.g., metabolic syndrome.

[0167] As used herein, "metabolic disorder" refers to any disease or disorder that disrupts normal metabolism, the process that converts food into energy at the cellular level. Metabolic disorders affect the ability of cells to carry out important biochemical reactions, including the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). Non-limiting examples of metabolic diseases include carbohydrate metabolism disorders, such as diabetes mellitus type I, diabetes mellitus type II, galactosemia, hereditary fructose intolerance, fructose 1,6-diphosphatase deficiency, glycogen storage disorders, inborn errors of glycosylation, insulin resistance, insulin deficiency, hyperinsulinemia, impaired glucose tolerance (IGT), abnormal glycogen metabolism; amino acid metabolism disorders, such as maple syrup urine disease (MSUD), or homocystinuria; organic acid metabolism disorders, such as methylmalonic aciduria, 3-methylglutaconic aciduria-Barth syndrome, glutaric aciduria, or 2-hydroxyglutaric aciduria-D and L types; disorders of fatty acid beta-oxidation, such as medium-chain acyl-CoA dehydrogenase deficiency (MCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (HCAAD), and 2-hydroxyglutaric aciduria-D and L types. lipid metabolism disorders, e.g., GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Fabry disease, Gaucher disease, Niemann-Pick disease, Krabbe disease, mucolipodosis, or mucopolysaccharidoses; mitochondrial disorders, e.g., mitochondrial cardiomyopathy; Leigh syndrome; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); myoclonus epilepsy with ragged-red fibers syndrome (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); Barth syndrome; peroxisomal disorders, e.g., Zellweger syndrome (cerebrohepatorenal syndrome), X-linked adrenoleukodystrophy, or Refsum disease.

[0168] In one embodiment, the metabolic disorder is metabolic syndrome.As used herein, the term "metabolic syndrome" refers to a disorder that includes clustering of components that reflect overnutrition, sedentary lifestyle, genetic factors, aging, and resulting excess obesity.Metabolic syndrome includes clustering of abnormal obesity, insulin resistance, hyperlipidemia, and elevated blood pressure, and is associated with other complications, including prothrombotic states, proinflammatory states, nonalcoholic fatty liver disease, and reproductive disorders.The prevalence of metabolic syndrome is high not only in the United States and the urbanized world, but also in developing countries.Metabolic syndrome is associated with approximately two times the risk of cardiovascular disease and five times the risk of incident type 2 diabetes.

[0169] Abdominal lipid accumulation (e.g., large waist circumference (high waist-to-hip ratio)), hypertension, insulin resistance, and hyperlipidemia play a central role in the metabolic syndrome and its individual components (e.g., central obesity, low fasting glucose (FBG) / prediabetes / diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension).

[0170] In one embodiment, the metabolic disorder is a carbohydrate metabolism disorder. In one embodiment, the carbohydrate metabolism disorder is diabetes.

[0171] As used herein, the term "diabetes" refers to a group of metabolic disorders characterized by high blood sugar (glucose) levels resulting from defects in insulin secretion or action, or both. There are two most common types of diabetes, primarily type 1 and type 2 diabetes, both resulting from the body's inability to control insulin. Insulin is a hormone released by the pancreas in response to elevated blood sugar (glucose) levels in the blood.

[0172] As used herein, the term "type I diabetes" refers to a chronic disease that occurs when the pancreas produces too little insulin to adequately control blood glucose levels. Type I diabetes is also called insulin-dependent diabetes, IDDM, and juvenile diabetes. People with type I diabetes (insulin-dependent diabetes) produce little or no insulin. About 6% of the U.S. population has some form of diabetes, but only about 10% of all diabetic patients have type I diabetes. Most people with type I diabetes develop the disorder before the age of 30. Type 1 diabetes represents the result of progressive autoimmune destruction of pancreatic beta cells, followed by insulin deficiency. More than 90% of the insulin-producing cells (beta cells) of the pancreas are permanently destroyed. The resulting insulin deficiency is severe, and people with type I diabetes must inject insulin regularly to survive.

[0173] In type II diabetes (also called non-insulin-dependent diabetes mellitus, NDDM), the pancreas continues to manufacture insulin, even at higher than normal levels. However, the body develops resistance to its effects, resulting in relative insulin deficiency. Type II diabetes can occur in children and adolescents, but it usually begins after age 30 and often develops gradually with age: about 15% of people over the age of 70 have type II diabetes. Obesity is a risk factor for type II diabetes, and 80 to 90 percent of people with the disorder are obese.

[0174] In some embodiments, diabetes includes prediabetes. "Prediabetes" refers to one or more early diabetic conditions, including impaired glucose utilization, impaired or impaired fasting blood glucose, impaired glucose tolerance, impaired insulin sensitivity and insulin resistance.Prediabetes is the main risk factor for the development of type 2 diabetes, cardiovascular disease and death.Many focus has been placed on developing therapeutic interventions that effectively treat prediabetes and thus prevent the development of type 2 diabetes.

[0175] Diabetes can be diagnosed by administering a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Primary examples of these categories include autoimmune diabetes, non-insulin-dependent diabetes mellitus (NDDM type 1), insulin-dependent diabetes mellitus (IDDM type 2), non-autoimmune diabetes, non-insulin-dependent diabetes mellitus (NIDDM type 2), and maturity-onset diabetes of the young (MODY). A further category, often called secondary, refers to diabetes resulting from some identifiable condition that causes or allows the development of a diabetic syndrome. Examples of secondary categories include diabetes caused by pancreatic disease, hormonal abnormalities, drug or chemical induced diabetes, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and diabetes of other causes. (See, for example, Harrison's (1996) 14th ed., New York, McGraw-Hill).

[0176] In one embodiment, metabolic disorder is dyslipidemia.As used herein, "dyslipidemia" or "dyslipidemia" refers to any disorder associated with or caused by the disruption of lipid metabolism.This term also includes any disorder, disease or condition that can result in hyperlipidemia, or a condition characterized by abnormal elevation of any or all lipids and / or lipoproteins in blood.This term also refers to genetic disorders, such as familial hypertriglyceridemia, familial partial lipodystrophy type 1 (FPLD1), or induced or acquired disorders, such as disorders induced or acquired as a result of disease, disorder or condition (e.g., renal failure), diet, or taking certain drugs (e.g., as a result of highly active antiretroviral therapy (HAART) used to treat AIDS or HIV).

[0177] Further examples of lipid metabolism disorders are atherosclerosis, hyperlipidemia, hypertriglyceridemia (drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergic blocker-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia. ), acute pancreatitis associated with hypertriglyceridemia, chylomicronemia, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), hyperlipidemia with heterogeneous LPL deficiency, hyperlipidemia with high LDL and heterogeneous LPL deficiency, fatty liver, or nonalcoholic steatohepatitis (NASH).

[0178] Cardiovascular diseases are also considered "metabolic disorders" as defined herein. These diseases can include coronary artery disease (also called ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, and stroke.

[0179] Weight-related disorders are also considered "metabolic disorders," as defined herein. Such disorders can include obesity, hypometabolic states, hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, sustained weight loss, or the risk of weight gain after weight loss.

[0180] Blood glucose disorder is further considered as "metabolic disorder" as defined herein. Such disorder can include diabetes, hypertension, and polycystic ovarian syndrome associated with insulin resistance. Other exemplary disorders of metabolic disorder can also include kidney transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogen storage disease type I, and Addison's disease.

[0181] In one embodiment, the INHBE-related disorder is primary hypertension. "Primary hypertension" is the result of environmental or genetic factors (e.g., the result of no apparent underlying medical cause).

[0182] In one embodiment, the INHBE-related disorder is secondary hypertension. "Secondary hypertension" has an identifiable underlying disorder that may be of multiple etiologies, including renal, vascular, and endocrine factors, such as renal parenchymal disease (e.g., polycystic kidney, glomerular or interstitial disease), renal vascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disorders (e.g., corticosteroid or mineralocorticoid excess, pheochromocytoma, hyper- or hypothyroidism, growth hormone excess, hyperparathyroidism), aortic stenosis, or oral contraceptive use.

[0183] In one embodiment, the INHBE-related disorder is resistant hypertension. "Resistant hypertension" is blood pressure that remains above target (e.g., systolic above 130 mmHg or diastolic above 90) despite the simultaneous use of three antihypertensive drugs of different classes, one of which is a thiazide diuretic. Subjects whose blood pressure is controlled by four or more medications are also considered to have resistant hypertension.

[0184] Additional diseases or conditions associated with metabolic disorders will be apparent to one of skill in the art and are within the scope of the present disclosure.

[0185] As used herein, a "therapeutically effective amount" is intended to include an amount of a modulator that, when administered to a subject having an INHBE-associated disorder, is sufficient to effect treatment of the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the modulator, how the agent is administered, the disease and its severity and medical history, age, weight, family history, genetic composition, type of prior or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0186] As used herein, a "prophylactically effective amount" is intended to include an amount of a molecule that, when administered to a subject with an INHBE-associated disorder, is sufficient to prevent or alleviate the disease or one or more symptoms of the disease. Alleviating the disease includes slowing the course of the disease or reducing the severity of later-onset disease. A "prophylactically effective amount" may vary depending on the modulator, how the modulator is administered, the degree of risk and medical history of the disease, age, weight, family history, genetic composition, type of prior or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0187] A "therapeutically effective amount" or "prophylactically effective amount" also includes that amount of a modulator that produces some desired effect, at a reasonable benefit / risk ratio applicable to any treatment. The modulators utilized in the methods of the invention may be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0188] As used herein, the phrase "pharmacologically acceptable" is used to refer to those compounds, materials, compositions, or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0189] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or steric acid), or a solvent encapsulant that participates in the transfer or transport of the subject compound from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

[0190] As used herein, the term "sample" includes collections of bodily fluids, cells, or tissues present within a subject, as well as similar bodily fluids, cells, or tissues isolated from a subject. Examples of bodily fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be from specific organs, parts of organs, or bodily fluids or cells within these organs. In certain embodiments, samples can be from the liver (e.g., the whole liver or a certain segment of the liver, or a certain type of cell within the liver, e.g., hepatocytes). In some embodiments, a "sample from a subject" refers to urine obtained from a subject. A "sample from a subject" can refer to blood from a subject, or serum or plasma from blood.

[0191] II. Modulators of the Invention The present invention provides modulators, i.e., inhibitors, of INHBE, and compositions comprising such modulators for use in modulating expression and / or activity of INHBE. In some embodiments, the modulators and compositions of the present invention are for use in treating a subject, e.g., a mammal, such as a human, susceptible to developing an INHBE-associated disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, dysglycemia, e.g., type II diabetes, prediabetes, dyslipidemia, e.g., hyperlipidemia, hypertension, cardiovascular disease, body weight disorder.

[0192] In one embodiment, the present invention provides a modulator of inhibin subunit beta E (INHBE).The modulator can be an oligonucleotide that targets INHBE, such as a double-stranded ribonucleic acid (dsRNA) or an antisense polynucleotide agent; an antibody that specifically binds to INHBE, or an antigen-binding fragment thereof, such as a monoclonal anti-INHBE antibody, or an antigen-binding fragment thereof; a small molecule; a guide RNA that performs ADAR editing, such as a guide RNA that comprises a stem-loop structure that binds to ADAR enzyme; or a guide RNA that performs CRISPR editing.

[0193] In one embodiment, a modulator of the invention is an RNAi, eg, double-stranded ribonucleic acid (dsRNA), agent that targets the INHBE gene.

[0194] In one embodiment, the modulator of the present invention is an antisense polynucleotide agent targeting the INHBE gene.

[0195] In one embodiment, a modulator of the invention is an antibody that specifically binds to INHBE, or an antigen-binding fragment thereof, such as a human, humanized or chimeric anti-INHBE antibody, or an antigen-binding fragment thereof.

[0196] In some embodiments, the modulator of INHBE is a small molecule.

[0197] In some embodiments, the modulator of INHBE is an aptamer. In some embodiments, the aptamer is an oligonucleotide aptamer. In some embodiments, the aptamer is a peptide aptamer.

[0198] In some embodiments, the modulator of INHBE is a guide RNA that performs double-stranded RNA-specific adenosine deaminase (ADAR) editing, e.g., a guide RNA that includes a stem-loop structure that binds to the ADAR enzyme.

[0199] In some embodiments, the modulator of INHBE is a guide RNA that performs CRIPR editing.

[0200] A. Oligonucleotides of the Invention Targeting INHBE i. iRNA of the invention In one embodiment, an oligonucleotide modulator of the invention targeting INHBE is an RNAi.

[0201] Thus, the present invention provides iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of inhibin subunit beta E (INHBE) gene. The gene can be in a cell, for example, in a cell in a subject, such as a human. The use of these iRNAs allows for targeted degradation of the mRNA of the corresponding gene (INHBE) in a mammal.

[0202] The iRNAs of the present invention are designed to target the human inhibin subunit beta E (INHBE) gene, including portions of the gene that are conserved in INHBE orthologs of other mammalian species. Without intending to be limited by theory, it is believed that combinations or subcombinations of the above characteristics and specific target sites or specific modifications in these iRNAs confer improved efficacy, stability, potency, durability, and safety to the iRNAs of the present invention.

[0203] Thus, the present invention provides methods for treating and preventing inhibin subunit beta E (INHBE) associated disorders, e.g., metabolic disorders, e.g., metabolic syndrome, carbohydrate metabolism disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertension, cardiovascular disease, body weight disorders, using iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the INHBE gene.

[0204] The iRNA of the present invention is at most about 30 nucleotides in length or less, for example, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, and comprises an RNA strand (antisense strand) having a region that is substantially complementary to at least a portion of the mRNA transcript of the INHBE gene.

[0205] In certain embodiments, one or both strands of a double-stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the INHBE gene. In some embodiments, such an iRNA agent having a long antisense strand can include a second RNA strand (sense strand) that is, e.g., 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0206] The use of iRNA of the present invention allows for targeted degradation of the mRNA of the corresponding gene (INHBE gene) in mammals. Using in vitro assays, the present inventors have demonstrated that iRNA targeting INHBE gene strongly mediates RNAi, resulting in significant inhibition of INHBE gene expression. Thus, methods and compositions comprising these iRNAs are useful for treating subjects with INHBE-related disorders, such as metabolic disorders, such as metabolic syndrome, carbohydrate metabolism disorders, such as type II diabetes, prediabetes, lipid metabolism disorders, such as hyperlipidemia, hypertension, cardiovascular disease, and body weight disorders.

[0207] Thus, the present invention provides methods and combination therapies for treating subjects with disorders that would benefit from inhibiting or reducing expression of the INHBE gene, e.g., inhibin subunit beta E (INHBE)-associated diseases, e.g., metabolic disorders, e.g., metabolic syndrome, carbohydrate metabolism disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertension, cardiovascular disease, body weight disorders, using iRNA compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the INHBE gene.

[0208] The present invention also provides a method for preventing at least one symptom in a subject having a disorder that would benefit from inhibiting or reducing expression of the INHBE gene, e.g., a metabolic disorder, e.g., metabolic syndrome, carbohydrate metabolism disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertension, cardiovascular disease, weight disorders.

[0209] In one aspect, the invention provides an iRNA that inhibits expression of the INHBE gene. In certain embodiments, the iRNA comprises a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of the INHBE gene in a cell, e.g., in a subject, a mammal, such as a human, susceptible to developing an INHBE-related disorder, e.g., a metabolic disorder, e.g., metabolic syndrome, carbohydrate metabolism disorders, e.g., type II diabetes, prediabetes, lipid metabolism disorders, e.g., hyperlipidemia, hypertension, cardiovascular disease, weight disorders. The dsRNAi agent comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed in the expression of the INHBE gene. The complementary region is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length).

[0210] When contacted with a cell expressing the INHBE gene, the iRNA inhibits the expression of the INHBE gene (e.g., human, primate, non-primate, or rat INHBE gene) by at least about 50%, as assayed by, for example, PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis using, for example, Western blot or flow cytometry. In certain embodiments, the inhibition of expression is determined by the qPCR method presented in the Examples herein, for example, for the siRNA at a concentration of 10 nM in a suitable biological cell or system, as provided in the Examples. In certain embodiments, the inhibition of expression in vivo is determined by knockdown of the human gene at the lowest level of RNA expression in a rodent expressing the human gene, for example, a mouse expressing the human target gene or an AAV-infected mouse, when administered, for example, as a single dose, for example, at 3 mg / kg.

[0211] dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary, generally completely complementary, to target sequence. Target sequence can be derived from the sequence of mRNA formed during expression of INHBE gene. The other strand (sense strand) comprises a region that is complementary to antisense strand, so that when combined under appropriate conditions, the two strands hybridize and form a duplex structure. As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to a complementary sequence on a separate oligonucleotide.

[0212] In general, the duplex structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-2 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex structure is 18-25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, e.g., 19-21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated as part of the present disclosure.

[0213] Similarly, the region complementary to the target sequence may be 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 1 9-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated as part of the present disclosure.

[0214] In some embodiments, the duplex structure is 19-30 base pairs in length. Similarly, the region complementary to the target sequence is 19-30 nucleotides in length.

[0215] In some embodiments, the dsRNA is about 19 to about 23 nucleotides long, or about 25 to about 30 nucleotides long. In general, the dsRNA is long enough to be used as a substrate for the Dicer enzyme. For example, it is known in the art that dsRNAs longer than about 21 to 23 nucleotides long can be used as substrates for Dicer. As those skilled in the art will also recognize, the region of an RNA targeted for cleavage is often quite often a part of a larger RNA molecule, which is an mRNA molecule. Where applicable, a "part" of an mRNA target is a continuous sequence of the mRNA target long enough to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0216] One of skill in the art will also recognize that a duplex region is the major functional portion of a dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, an RNA molecule or complex of RNA molecules having a duplex region of more than 30 base pairs that targets a desired RNA for cleavage, e.g., is a dsRNA, so long as it is processed into a functional duplex of 15-30 base pairs. Thus, one skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting expression of the INHBE gene is not generated by cleavage of a large dsRNA in the target cell.

[0217] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, for example, 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhangs may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) overhang may be on the 5'-end, the 3'-end, or both ends of the antisense strand or the sense strand of the dsRNA.

[0218] dsRNA can be synthesized by standard methods known in the art. The double-stranded RNAi compounds of the present invention can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the strand components are annealed. The individual strands of the siRNA compounds can be prepared using liquid-phase organic synthesis or solid-phase organic synthesis, or both. Organic synthesis has the advantage that the oligonucleotide strands containing non-natural nucleotides or modified nucleotides can be easily prepared. Similarly, the single-stranded oligonucleotides of the present invention can be prepared using liquid-phase organic synthesis or solid-phase organic synthesis, or both.

[0219] In one embodiment, the dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the sequence group presented in any one of Tables 2-3, and the corresponding antisense strand of the sense strand is selected from the sequence group of any one of Tables 2-3. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the mRNA generated upon expression of the INHBE gene. Thus, in this embodiment, the dsRNA will comprise two oligonucleotides, one oligonucleotide described as the sense strand in any one of Tables 2-3, and the second oligonucleotide described as the corresponding antisense strand of the sense strand in any one of Tables 2-3.

[0220] In certain embodiments, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides, hi other embodiments, the substantially complementary sequences of the dsRNA are contained in a single oligonucleotide.

[0221] For example, the sequences in Table 3 are not described as modified or conjugated sequences, but it will be understood that the RNA of the present disclosure, e.g., the dsRNA of the present invention, can include any one of the sequences set forth in any one of Tables 2-3, unmodified, unconjugated, or modified or conjugated in a manner different from that described therein. In other words, the present invention encompasses the dsRNAs of Tables 2-3, unmodified, unconjugated, or modified or conjugated as described herein.

[0222] Those skilled in the art are aware that dsRNAs having a duplex structure of about 20-23 base pairs, for example 21 base pairs, are said to be particularly effective in inducing RNA interference [Elbashir et al., EMBO 2001, 20:6877-6888]. However, other researchers have found that shorter duplex structures or longer RNA duplex structures can also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the above-described embodiments, due to the nature of the oligonucleotide sequences presented in any one of Tables 2-3, the dsRNAs described herein may include at least one strand with a length of at least 21 nucleotides. It may be reasonably expected that shorter duplexes having any one of the sequences in any one of Tables 2-3, minus only a few nucleotides at one or both ends, may be similarly effective compared to the dsRNAs described above.Thus, it is contemplated that dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides derived from any one of the sequences in any one of Tables 2-3, and differing in their ability to inhibit expression of the INHBE gene by no more than about 5, 10, 15, 20, 25, or 30% inhibition of a dsRNA containing the full-length sequence, are within the scope of the present invention.

[0223] In addition, the RNAs presented in Tables 2-3 identify a site(s) in the INHBE transcript that is susceptible to RISC-mediated cleavage. Thus, the present invention further features an iRNA that targets within one of these sites. As used herein, an iRNA is said to target within a specific site of an RNA transcript if the iRNA promotes cleavage of the transcript at any position within this specific site. Such an iRNA generally comprises at least about 19 contiguous nucleotides from any one of the sequences presented in any one of Tables 2-3 coupled to additional nucleotide sequences taken from regions adjacent to the selected sequence within the INHBE gene.

[0224] ii. Antisense polynucleotide agents of the invention In one embodiment, the modulator of the invention is an antisense polynucleotide agent.

[0225] Thus, the present invention provides polynucleotide agents, e.g., antisense polynucleotide agents, that target the INHBE gene and inhibit expression of the INHBE gene, and compositions comprising such agents. In one embodiment, the polynucleotide agents, e.g., antisense polynucleotide agents, inhibit expression of the INHBE gene in a cell, e.g., in a cell in a mammalian subject, such as a human, having an INHBE-associated disorder, e.g., acromegaly, gigantism, or cancer.

[0226] Polynucleotide agents, e.g., antisense polynucleotide agents, of the invention include a region of complementarity that is complementary to at least a portion of an mRNA formed upon expression of the INHBE gene. The region of complementarity can be about 50 nucleotides or less in length (e.g., 22-12, 20-14, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides or less in length). When contacted with a cell expressing the INHBE gene, the antisense polynucleotide agent inhibits expression of the INHBE gene (e.g., a human, primate, non-primate, or avian INHBE gene) by at least 20%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as, for example, by immunofluorescence analysis using Western blot or flow cytometry. In a preferred embodiment, inhibition of expression is determined at a 10 nM concentration using the cell line and delivery method.

[0227] The region of complementarity between an antisense polynucleotide agent and a target sequence can be substantially complementary (e.g., there is sufficient complementarity between an antisense polynucleotide agent and a target nucleic acid so that they specifically hybridize and induce a desired effect), but is generally completely complementary to the target sequence. The target sequence can be derived from the sequence of the mRNA formed upon expression of the INHBE gene.

[0228] Thus, in one embodiment, the antisense polynucleotide agent of the present invention specifically hybridizes to a target nucleic acid molecule, e.g., an mRNA encoding INHBE, and comprises a contiguous nucleotide sequence that corresponds to the reverse complement of the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9, or a fragment of any one of SEQ ID NOs: 1, 3, 5, 7, or 9.

[0229] In some embodiments, the antisense polynucleotide agent of the present invention can be substantially complementary to a target sequence. For example, the antisense polynucleotide agent that is substantially complementary to a target sequence can comprise a contiguous nucleotide sequence that contains 5 or less mismatches (e.g., 1 or less, 2 or less, 3 or less, 4 or less, or 5 or less mismatches) when hybridizing with a target sequence, for example, a corresponding region of a nucleic acid that codes for a mammalian INHBE mRNA. In some embodiments, the contiguous nucleotide sequence contains 1 or less mismatches when hybridizing with a target sequence, for example, a corresponding region of a nucleic acid that codes for a mammalian INHBE mRNA.

[0230] In some embodiments, an antisense polynucleotide agent of the invention that is substantially complementary to a target sequence comprises a contiguous nucleotide sequence that is at least 80% complementary, such as at least 85%, 90%, 95%, or 100% complementary, over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9, or a fragment of any one of SEQ ID NOs: 1, 3, 5, 7, or 9.

[0231] In some embodiments, an antisense polynucleotide agent comprises a contiguous nucleotide sequence that is fully complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1, 3, 5, 7, or 9 (or a fragment of any one of SEQ ID NOs: 1-5). For example, the nucleotide sequence of the antisense polynucleotide agent is fully complementary over its entire length to the equivalent region of nucleotides 1-20 of GenBank Accession No. NM_031479.5 (SEQ ID NO: 1) (see, e.g., Tables 4 or 5).

[0232] Antisense polynucleotide agents can be about 4-50 nucleotides in length, or, for example, about 8-49, 8-48, 8-47, 8-46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 10-49 , 10~48, 10~47, 10~46, 10~45, 10~44, 10~43, 10~42, 10~41, 10~40, 10~39, 10~38, 10~37, 10~36, 10~35, 10~34, 10~33, 10~32, 10~31, 10~30, 10~29, 10~2 8, 10~27, 10~26, 10~25, 10~24, 10~23, 10~22, 10~21, 10~20, 10~19, 10~18, 10~17, 10~16, 10~15, 10~14, 10~13, 10~12, 10~11, 11~49, 11~48, 11~47, 11~ 46, 11~45, 11~44, 11~43, 11~42, 11~41, 11~40, 11~39, 11~38, 11~37, 11~36, 11~35, 11~34, 11~33, 11~32, 11~31, 11~30, 11~29, 11~28, 11~27, 11~26, 11 ~25, 11~24, 11~23, 11~22, 11~21, 11~20, 11~19, 11~18, 11~17, 11~16, 11~15, 11~14, 11~13, 11~12, 12~49, 12~48, 12~47, 12~46, 12~45, 12~44, 12~43, 1 2~42, 12~41, 12~40, 12~39, 12~38, 12~37, 12~36, 12~35, 12~34, 12~33, 12~32, 12~31, 12~30, 12~29, 12~28, 12~27, 12~26, 12~25, 12~24, 12~23, 12~22, 12~21, 12~20, 12~19, 12~18, 12~17, 12~16, 12~15, 12~14, 12~13, 13~49, 13~48, 13~47, 13~46, 13~45, 13~44, 13~43, 13~42, 13~41, 13~40, 13~39, 13~38,13~37、13~36、13~35、13~34、13~33、13~32、13~31、13~30、13~29、13~28、13~27、13~26、13~25、13~24、13~23、13~22、13~21、13~20、13~19、13~18、13~17、13~16、13~15、13~14、14~49、14~48、14~47、14~46、14~45、14~44、14~43、14~42、14~41、14~40、14~39、14~38、14~37、14~36、14~35、14~34、14~33、14~32、14~31、14~30、14~29、14~28、14~27、14~26、14~25、14~24、14~23、14~22、14~21、14~20、14~19、14~18、14~17、14~16、14~15、15~49、15~48、15~47、15~46、15~45、15~44、15~43、15~42、15~41、15~40、15~39、15~38、15~37、15~36、15~35、15~34、15~33、15~32、15~31、15~30、15~29、15~28、15~27、15~26、15~25、15~24、15~23、15~22、15~21、15~20、15~19、15~18、15~17、15~16、16~49、16~48、16~47、16~46、16~45、16~44、16~43、16~42、16~41、16~40、16~39、16~38、16~37、16~36、16~35、16~34、16~33、16~32、16~31、16~30、16~29、16~28、16~27、16~26、16~25、16~24、16~23、16~22、16~21、16~20、16~19、16~18、16~17、17~49、17~48、17~47、17~46、17~45、17~44、17~43、17~42、17~41、17~40、17~39、17~38、17~37、17~36、17~35、17~34、17~33、17~32、17~31、17~30、17~29、17~28、17~27、17~26、17~25、17~24、17~23、17~22、17~21、17~20、17~19、17~18、18~49、18~48、18~47、18~46、18~45、18~44、18~43、18~42、18~41、18~40、18~39、18~38、18~37、18~36、18~35、18~34、18~33、18~32、18~31、18~30、18~29、18~28、18~27、18~26、18~25、18~24、18~23、18~22、18~21、18~20、19~49、19~48、19~47、19~46、19~45、19~44、19~43、19~42、19~41、19~40、19~39、19~38、19~37、19~36、19~35、19~34、19~33、19~32、19~31、19~30、19~29、19~28、19~27、19~26、19~25、19~24、19~23、19~22、19~21、19~20、20~49、20~48、20~47、20~46、20~45、20~44、20~43、20~42、20~41、20~40、20~39、20~38、20~37、20~36、20~35、20~34、20~33、20~32、20~31、20~30、20~29、20~28、20~27、20~26、20~25、20~24、20~23、20~22、20~21、21~49、21~48、21~47、21~46、21~45、21~44、21~43、21~42、21~41、21~40、21~39、21~38、21~37、21~36、21~35、21~34、21~33、21~32、21~31、21~30、21~29、21~28、21~27、21~26、21~25、21~24、21~23、21~22、22~49、22~48、22~47、22~46、22~45、22~44、22~43、22~42、22~41、22~40、22~39、22~38、22~37、22~36、22~35、22~34、22~33、22~32、22~31、22~30、22~29、22~28、22~27、22~26、22~25、22~24、22~23、23~49、23~48、23~47、23~46、23~45、23~44、23~43、23~42、23~41、23~40、23~39、23~38、23~37、23~36、23~35、23~34、23~33、23~32、23~31、23~30、23~29、23~28、23~27、23~26、23~25、23~24、24~49、24~48、24~47、24~46、24~45, 24~44, 24~43, 24~42, 24~41, 24~40, 24~39, 24~38, 24~37, 24~36, 24~35, 24~34, 24~33, 24~32, 24~31, 24~30, 24~29, 24~28, 24~27, 24~26, 24~25 , 25~49, 25~48, 25~47, 25~46, 25~45, 25~44, 25~43, 25~42, 25~41, 25~40, 25~39, 25~38, 25~37, 25~36, 25~35, 25~34, 25~33, 25~32, 25~31, 25~30, 25~2 9, 25-28, 25-27, 25-26, 26-49, 26-48, 26-47, 26-46, 26-45, 26-44, 26-43, 26-42, 26-41, 26-40, 26-39, 26-38, 26-37, 26-36, 26-35, 26-34, 26-33, 26- 32, 26-31, 26-30, 26-29, 26-28, 26-27, 27-49, 27-48, 27-47, 27-46, 27-45, 27-44, 27-43, 27-42, 27-41, 27-40, 27-39, 27-38, 27-37, 27-36, 27-35, 27 ~34, 27~33, 27~32, 27~31, 27~30, 27~29, 27~28, 28~49, 28~48, 28~47, 28~46, 28~45, 28~44, 28~43, 28~42, 28~41, 28~40, 28~39, 28~38, 28~37, 28~36, 2 8~35, 28~34, 28~33, 28~32, 28~31, 28~30, 28~29, 29~49, 29~48, 29~47, 29~46, 29~45, 29~44, 29~43, 29~42, 29~41, 29~40, 29~39, 29~38, 29~37, 29~36, Any subrange within the length of 29-35, 29-34, 29-33, 29-32, 29-31, 29-30, 30-49, 30-48, 30-47, 30-46, 30-45, 30-44, 30-43, 30-42, 30-41, 30-40, 30-39, 30-38, 30-37, 30-36, 30-35, 30-34, 30-33, 30-32, or 30-31 nucleotides, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,It may comprise a contiguous nucleotide sequence of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0233] In some embodiments, the antisense polynucleotide agent may comprise a contiguous nucleotide sequence of 22 nucleotides or less, for example, 21 nucleotides, 20 nucleotides, 19 nucleotides, 18 nucleotides, 17 nucleotides, 16 nucleotides, 15 nucleotides, or 14 nucleotides or less.In other embodiments, the antisense polynucleotide agent of the present invention is 20 nucleotides in length.In other embodiments, the antisense polynucleotide agent of the present invention is 14 nucleotides in length.In certain embodiments, the polynucleotide is at least 12 nucleotides in length.

[0234] In one embodiment, an antisense polynucleotide agent of the invention comprises a sequence selected from the sequences presented in Table 4 or Table 5. Although the sequences in Table 5 are set forth as modified or conjugated sequences, it will be understood that an antisense polynucleotide agent of the invention can also comprise any one of the sequences set forth in Table 5 that is unmodified, unconjugated, or modified or conjugated differently than those set forth therein.

[0235] Due to the nature of the nucleotide sequences presented in Tables 4 or 5, the antisense polynucleotide agents of the present invention may comprise one of the sequences of Tables 3 or 5, lacking only a few nucleotides at one or both ends, and still remain similarly effective compared to the antisense polynucleotide agents described above. Thus, antisense polynucleotide agents having a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides derived from one of the sequences of Tables 4 or 5, and differing in their ability to inhibit the expression of the INHBE gene from antisense polynucleotides comprising the full-length sequence by no more than 5, 10, 15, 20, 25, or 30% inhibition, are considered to be within the scope of the present invention. Furthermore, the antisense polynucleotide agents presented in Tables 4 and 5 identify a region(s) in the INHBE transcript that is susceptible to antisense inhibition (e.g., the region encompassed by the start and stop positions compared to the nucleotide sequences of Table 4). As such, the present invention further features antisense polynucleotide agents that target within one of these sites.

[0236] As used herein, an antisense polynucleotide agent is said to target within a specific site of RNA transcript if it promotes the antisense inhibition of target at that site.Such antisense polynucleotide agent usually comprises at least 14 consecutive nucleotides from one of the sequences listed in Table 4 or 5, combined with additional nucleotide sequence taken from the region following the selected sequence in INHBE gene.

[0237] Target sequences are typically 4-50 nucleotides in length, although there is wide variation in the suitability of particular sequences within this range to direct antisense inhibition of any given target RNA. The various software packages and guidelines presented herein provide guidance for the identification of optimal target sequences for any given gene target, but empirical approaches can also be used in which a "window" or "mask" of a given size (20 nucleotides, as a non-limiting example) is placed, either verbatim or figuratively (including, for example, in silico), over the target RNA sequence to identify sequences within the size range that can be used as target sequences. By gradually moving the sequence "window" one nucleotide upstream or downstream of the initial sequence position, the next potential target sequence can be identified until a complete set of possible sequences has been identified for any given target size selected. This process, combined with the systematic synthesis and testing of identified sequences (using the assays described herein or known in the art) to identify those sequences that perform optimally, can identify those RNA sequences that mediate the best inhibition of target gene expression when targeted by antisense polynucleotide agents.Thus, for example, the sequences identified in Table 4 or 5 represent effective target sequences, but it is believed that further optimization of antisense inhibition efficiency can be achieved by gradually "walking the window" one nucleotide upstream or downstream of a given sequence to identify sequences that have equal or better inhibition characteristics.

[0238] Furthermore, for any sequence identified in, for example, Table 4 or 5, further optimization could be achieved by either systematically adding or removing nucleotides to generate longer or shorter sequences, and testing the sequences generated by walking the window that makes the target RNA longer or shorter in size from that point. This approach could also be combined with generating new candidate targets by testing the effectiveness of antisense polynucleotide agents based on those target sequences in inhibition assays known in the art or described herein, resulting in further improvements in the efficiency of inhibition. Furthermore, such optimized sequences could be adjusted, for example, by introducing modified nucleotides described herein or known in the art, adding or changing length, or other modifications known in the art or discussed herein, to further optimize the molecule as an expression inhibitor (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

[0239] iii. Modified Oligonucleotides of the Invention In certain embodiments, the oligonucleotide of the present invention, for example, dsRNA agent or antisense polynucleotide agent, is unmodified and does not include chemical modifications or conjugations known in the art and described herein.In other embodiments, the oligonucleotide of the present invention, for example, dsRNA agent or antisense polynucleotide agent, is chemically modified to enhance stability or other beneficial characteristics.In certain embodiments of the present invention, substantially all of the nucleotides of the oligonucleotide of the present invention, for example, dsRNA agent or antisense polynucleotide agent, are modified.In other embodiments of the present invention, substantially all of the nucleotides of the oligonucleotide, for example, dsRNA agent or antisense polynucleotide agent, or substantially all of the nucleotides of the oligonucleotide, for example, dsRNA agent or antisense polynucleotide agent, are modified, i.e., no more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in the chain of the oligonucleotide, for example, dsRNA agent or antisense polynucleotide agent.

[0240] Nucleic acids featured in the present invention can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkages) or 3'-end modifications (conjugation, DNA nucleotide inverted linkages, etc.); base modifications, such as replacement with a stabilizing base, an unstable base, or a base that base pairs with an expanded repertoire of partners, removal of a base (abasic nucleotide), or a conjugated base; sugar modifications (e.g., at the 2' or 4' position) or replacement of a sugar; or backbone modifications, including modification or replacement of a phosphodiester linkage. Specific examples of oligonucleotide compounds useful in the embodiments described herein include, but are not limited to, oligonucleotides containing modified backbones, such as RNA, or oligonucleotides that do not contain natural internucleoside linkages, such as RNA. The oligonucleotide with modified backbone, for example, RNA, includes, among others, RNA that does not have phosphorus atom in backbone.For the purpose of this specification, and as sometimes referred to in the art, the modified oligonucleotide with no phosphorus atom in their internucleoside backbone, for example, RNA, can also be considered as oligonucleoside.In some embodiments, the modified oligonucleotide will have phosphorus atom in their internucleoside backbone.

[0241] Modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, their 2'-5' linked analogs, and analogs with reverse polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salt forms, mixed salt forms, and free acid forms are also included.In some embodiments of the present invention, the oligonucleotides of the present invention, such as dsRNA agents or antisense polynucleotide agents, are in free acid form. In other embodiments of the present invention, the oligonucleotide, for example, dsRNA agent or antisense polynucleotide agent, is in salt form. In one embodiment, the oligonucleotide, for example, dsRNA agent or antisense polynucleotide agent, of the present invention is in sodium salt form. In certain embodiments, when the oligonucleotide, for example, dsRNA agent or antisense polynucleotide agent, of the present invention is in sodium salt form, sodium ions are present in the agent as counterions to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent. The oligonucleotide in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterions comprises no more than 5, 4, 3, 2, or 1 phosphodiester and / or phosphorothioate linkages without sodium counterions. In some embodiments, when the oligonucleotide, for example, dsRNA agent or antisense polynucleotide agent, of the present invention is in sodium salt form, sodium ions are present in the oligonucleotide as counterions to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0242] Representative United States patents which teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,277,223; and 5,281,636, the entire contents of each of which are incorporated herein by reference. No. 78,302; No. 5,286,717; No. 5,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; Same No. 5,466,677; Same No. 5,476,925; Same No. 5,519,126; Same No. 5,536,821; Same No. 5,541,316; Same No. 5,550,111; Same No. 5,563,2 No. 53; No. 5,571,799; No. 5,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6, No. 169,170; No. 6,172,209; No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423 ;6,531,590;6,534,639;6,608,035;6,683,167;6,858,715;6,867,294;6,878,805;7,015,315;7,041,816;7,273,933;7,321,029; and U.S. Reissue Patent No. 39464, among others.

[0243] Modified oligonucleotide backbones, such as RNA backbones, in which there is no phosphorus atom, have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or internucleoside linkages with one or more short chains of heteroatoms or heterocycles.Modified oligonucleotide backbones include modified RNA backbones with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and mixed N, O, S, and CH. 2 It comprises a backbone having constituent parts.

[0244] Representative United States patents which teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466, 677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0245] RNA mimics suitable for use in the oligonucleotides presented herein, such as dsRNA agents or antisense polynucleotide agents, are envisioned in which both the sugar and internucleoside linkages, i.e., backbone, of nucleotide units are replaced by novel groups. Base units are maintained for hybridization with appropriate nucleic acid targeting compounds. One such oligomeric compound, in which RNA mimics have been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleobases are retained and are directly or indirectly bound to the aza nitrogen atom of the backbone's amide portion. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the oligonucleotides of the invention, such as iRNA, are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0246] Some embodiments featured in the present invention include oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly the -CH 2 -NH-CH 2 -, -CH 2 -N(CH 3 )-O-CH 2 -[known as the methylene (methylimino) backbone or MMI backbone], -CH 2 -ON(CH 3 )-CH 2 -, -CH 2 -N(CH 3 )-N(CH 3 )-CH 2 - and -N(CH 3 )-CH2 -CH 2 -, and the amide backbone of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506. The natural phosphodiester backbone can be represented as OP(O)(OH)-OCH2-.

[0247] Modified oligonucleotides may also contain one or more substituted sugar moieties. Oligonucleotides, such as dsRNA agents or antisense polynucleotide agents featured herein, may contain at the 2' position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted, C 1 ~C 10 Alkyl of, or C 2 ~C 10 Exemplary suitable modifications include one of the following: O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are from 1 to about 10. In another embodiment, the dsRNA comprises at the 2' position: 1 ~C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalating agent, group for improving the pharmacokinetic properties of an oligonucleotide, or group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. In some embodiments, the modification is 2'-methoxyethoxy [2'-O-CH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE]. 2 CH 2 OCH 3 ] (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification includes 2'-dimethylaminooxyethoxy, i.e., O(CH 2 ) 2 ON(CH 3 ) 2 groups, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers within these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0248] Other modifications include 2'-methoxy (2'-OCH 3 ), 2'-aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide, or in 2'-5' linked dsRNAs, and at the 5' position of the 5' terminal nucleotide. Oligonucleotides, such as dsRNA agents or antisense polynucleotide agents, can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents that teach such modified sugar structures include U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,515,786; and 5,516,787, all of which are co-owned with this application. Nos. 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, the entire contents of each of the foregoing being incorporated herein by reference.

[0249] Oligonucleotides, such as dsRNA agents or antisense polynucleotide agents, can also include modifications or substitutions of nucleobases (often referred to in the art simply as "bases").As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-haloadenines and guanines, 8-aminoadenine and guanine, 8-thioladenine and 8-thioladenine, and 8-thioladenine. and other synthetic and natural nucleobases such as -thiolguanine, 8-thioalkyladenines and guanines, 8-hydroxyl adenine and analogues, other 8-substituted adenines and guanines, 5-halouracils and cytosines, particularly 5-bromouracil and cytosine, 5-trifluoromethyluracil and cytosine, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention.These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2 substituted purines, N-6 substituted purines, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is an exemplary base substitution, even more particularly when combined with 2'-O-methoxyethyl sugar modification.

[0250] Representative United States patents which teach the preparation of certain of the above-referenced modified nucleobases, as well as other modified nucleobases, include the above-referenced U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,706; and, more particularly, U.S. Pat. Nos. 3,687,808, 3,687,808, 3,687,808, 3,787,808, 3,845,205, 3,925,706, and 3,102,177. No. 11; No. 5,552,540; No. 5,587,469; No. 5,594,121; No. 5,596,091; No. 5,614,617 No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; No. 6,166,197; Including, but not limited to, Nos. 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088.

[0251] In some embodiments, the oligonucleotides, e.g., dsRNA agents or antisense polynucleotide agents of the present disclosure, may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified with a ring formed by bridging two adjacent or non-adjacent carbons. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a ring formed by bridging two adjacent or non-adjacent carbons of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4' and 2' carbons of the sugar ring, optionally through a 2' acyclic oxygen atom. Thus, in some embodiments, the agents of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety that includes an additional bridge connecting the 2' and 4' carbons of the ribose moiety. In other words, an LNA is a nucleoside having a 4'-CH2 A bicyclic nucleoside is a nucleotide that contains a bicyclic sugar moiety that contains an -O-2' bridge. This structure effectively "locks" the ribose in a 3' internal structure conformation. The addition of a locked nucleic acid to an siRNA has been shown to increase the stability of the siRNA in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193]. Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides that contain a bridge between the 4' and 2' atoms of the ribosyl ring. In certain embodiments, an antisense polynucleotide agent of the invention comprises one or more bicyclic nucleosides that include a 4'-2' bridge.

[0252] Locked nucleosides have the structure (stereochemistry omitted):

[0253] [ka] where B is a nucleobase or modified nucleobase and L is a linking group connecting the 2' carbon to the 4' carbon of the ribose ring. An example of such a 4'-2' bridged bicyclic nucleoside is 4'-(CH 2 )-O-2'(LNA);4'-(CH 2 )-S-2';4'-(CH 2 ) 2 -O-2'(ENA);4'-CH(CH 3 )-O-2' (also known as "constrained ethyl" or "cEt"), and 4'-CH(CH 2 OCH 3 )-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH 3 )(CH 3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH 2 -N(OCH 3 )-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH 2 -ON(CH 3 )-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH 2 -N(R)-O-2', where R is H, C1-C12 alkyl, or a nitrogen protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH 2 -C(H)(CH 3 )-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH 2 -C(=CH 2 )-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426), the entire contents of each of the foregoing are incorporated herein by reference.

[0254] Additional representative U.S. patents and U.S. patent publications which teach the preparation of locked nucleic acid nucleotides include the following, the entire contents of each of which are incorporated herein by reference: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034, 133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US2008 / 0039618; and US2009 / 0012281.

[0255] For example, any of the foregoing bicyclic nucleosides can be prepared having one or more sugar stereochemical configurations (see WO 99 / 14226), including α-L-ribofuranose and β-D-ribofuranose.

[0256] The nucleotides of an oligonucleotide, e.g., a dsRNA agent or an antisense polynucleotide agent, can also be modified to include one or more constrained ethyl nucleotides. As used herein, "constrained ethyl nucleotide" or "cEt" refers to a 4'-CH(CH 3 )-O-2' bridge (i.e., L in the structure above). In one embodiment, the constrained ethyl nucleotide is in the S conformation, referred to herein as "S-cEt."

[0257] The oligonucleotide of the present invention, for example, dsRNA agent or antisense polynucleotide agent, can also include one or more "conformationally locked nucleotides" ("CRN"). A CRN is a nucleotide analog with a linker that connects the C2' carbon of ribose with the C4' carbon or the C3 carbon of ribose with the -C5' carbon. The CRN locks the ribose ring into a stable conformation and increases the hybridization affinity to mRNA. The linker is long enough to place oxygen in the optimal position for stability and affinity, which results in reduced puckering of the ribose ring.

[0258] Representative publications that teach the preparation of certain of the CRNs mentioned above include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383; and PCT Publication No. WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0259] In some embodiments, the oligonucleotides, e.g., dsRNA agents or antisense polynucleotide agents of the present disclosure, comprise one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked non-cyclic nucleic acids, in which any of the sugar bonds are removed to form an unlocked "sugar" residue. In one example, UNAs also encompass monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed [see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference].

[0260] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent Publication Nos. 8,314,227; and 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0261] Potential stabilizing modifications for the termini of oligonucleotide molecules, e.g., RNA molecules, may include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT (idT) and other stabilizing modifications. Disclosure of this modification may be found in PCT Publication No. WO2011 / 005861.

[0262] Other modifications to the nucleotides of, for example, dsRNA agents or antisense polynucleotide agents of the present invention include 5' phosphate or 5' phosphate mimics, for example, 5' terminal phosphate or phosphate mimics, on the antisense iRNA strand.Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0263] iv. Modified iRNAs Containing the Motifs of the Invention In certain aspects of the present invention, the double-stranded RNA agent of the present invention comprises agents with chemical modifications, for example, as disclosed in WO2013 / 075035, the entire contents of each of which are incorporated herein by reference.One or more motifs of three identical modifications on three consecutive nucleotides as shown herein and in WO2013 / 075035 can be introduced into the sense or antisense strand of dsRNAi agent, particularly at or near the cleavage site.In some embodiments, the sense and antisense strands of dsRNAi agent can be completely modified in other ways.The introduction of these motifs, if present, interrupts the modification pattern of the sense or antisense strand.The dsRNAi agent can be conjugated with GalNAc derivative ligand, for example, on the sense strand.

[0264] More specifically, gene silencing activity of a dsRNAi agent was observed when the sense and antisense strands of the double-stranded RNA agent were fully modified to have one or more motifs with three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0265] Thus, the present invention provides double-stranded RNA agents capable of inhibiting expression of a target gene (i.e., an INHBE gene) in vivo. The RNAi agent includes a sense strand and an antisense strand. Each strand of the RNAi agent can be, for example, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0266] The sense strand and the antisense strand typically form a double stranded, double stranded RNA ("dsRNA"), also referred to herein as a "dsRNAi agent". The duplex region of the dsRNAi agent can be, for example, 27-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0267] In certain embodiments, the dsRNAi agent may contain one or more overhanging regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be independently 1-6 nucleotides long, e.g., 2-6 nucleotides long, 1-5 nucleotides long, 2-5 nucleotides long, 1-4 nucleotides long, 2-4 nucleotides long, 1-3 nucleotides long, 2-3 nucleotides long, or 1-2 nucleotides long. In certain embodiments, the overhanging region may include the extended overhanging region presented above. The overhang may be the result of one strand being longer than the other strand, or the result of two strands of the same length being cohesive end strands. The overhang may form a mismatch with the target mRNA, may be complementary to the gene sequence being targeted, or may be another sequence. The first and second strands may also be connected by additional bases, e.g., to form a hairpin, or may be connected by other non-basic linkers.

[0268] In certain embodiments, the nucleotides in the overhang region of a dsRNAi agent can each independently be modified or unmodified nucleotides, including, but not limited to, 2' sugar modified nucleotides, such as 2'-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0269] For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, can be complementary to the targeted gene sequence, or can be a different sequence.

[0270] The 5'-overhang or 3'-overhang on the sense strand, antisense strand, or both strands of the dsRNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides with phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In some embodiments, the 3'-overhang is present in the antisense strand. In some embodiments, the 3'-overhang is present in the sense strand.

[0271] dsRNAi agent can contain only one overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, single-stranded overhang can be located at the 3' end of sense strand, or alternatively, at the 3' end of antisense strand.RNAi can also have a blunt end located at the 5' end of antisense strand (i.e., the 3' end of sense strand), or vice versa.Generally, the antisense strand of dsRNAi agent has a nucleotide overhang at the 3' end, and the 5' end is blunt.Without wishing to be bound by theory, the asymmetry between the blunt end at the 5' end of antisense strand and the 3' end overhang of antisense strand is favorable for the loading of guide strand into RISC process.

[0272] In certain embodiments, the dsRNAi agent is 19 nucleotides long and both blunt-ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end.The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0273] In other embodiments, the dsRNAi agent is 20 nucleotides in length and both blunt-ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 of the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0274] In yet another embodiment, the dsRNAi agent is 21 nucleotides in length and both blunt-ended, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 of the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 of the 5' end.

[0275] In certain embodiments, dsRNAi agent comprises a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, wherein the sense strand comprises at least one of the following motifs: three 2'-F modifications on three consecutive nucleotides at the 9th, 10th and 11th positions of the 5' end; the antisense strand comprises at least one of the following motifs: three 2'-O-methyl modifications on three consecutive nucleotides at the 11th, 12th and 13th positions of the 5' end, and one end of the RNAi agent is blunt, while the other end comprises a 2-nucleotide overhang.In one embodiment, the 2-nucleotide overhang is at the 3' end of the antisense strand.

[0276] When a two nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhanging nucleotides and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In certain embodiments, every nucleotide in the sense strand and the antisense strand of the dsRNAi agent, including a nucleotide that is part of a motif, is a modified nucleotide. In certain embodiments, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. The dsRNAi agent is a ligand (GalNAc 3 etc.) may be further included.

[0277] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, the sense strand being 25-30 nucleotide residues in length, beginning with the 5'-terminal nucleotide (position 1), and positions 1-23 of the first strand comprising at least 8 ribonucleotides; the antisense strand being 36-66 nucleotide residues in length, beginning with the 3'-terminal nucleotide, and comprising at least 8 ribonucleotides at positions 1-23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired from the sense strand, and up to 6 contiguous nucleotides at the 3'-end are unpaired from the sense strand, thereby forming a single-stranded 3' overhang of 1-6 nucleotides; and the 5'-end of the antisense strand being unpaired from the sense strand, and comprising 10-30 contiguous nucleotides. the sense strand comprises consecutive nucleotides, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantially duplexed region between the sense strand and the antisense strand; the antisense strand is sufficiently complementary to the target RNA over a length of at least 19 ribonucleotides of the antisense strand such that the double-stranded nucleic acid reduces expression of the target gene when introduced into a mammalian cell; the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of the motifs is at or near the cleavage site. The antisense strand contains at least one motif of 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site of the strand.

[0278] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, the dsRNAi agent comprising a first strand that is at least 25 nucleotides and up to 29 nucleotides in length, and a second strand that is up to 30 nucleotides in length with at least one of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 of the 5' end; the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand has a duplex region at its 3' end that is 1-4 nucleotides longer than the first strand and is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA over a length of at least 19 nucleotides of the second strand such that when the RNAi agent is introduced into a mammalian cell, it reduces expression of the target gene, where cleavage of the dsRNAi agent by Dicer results in an siRNA that includes the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. The dsRNAi agent may further comprise a ligand.

[0279] In certain embodiments, the sense strand of a dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs is present at the cleavage site in the sense strand.

[0280] In certain embodiments, the antisense strand of a dsRNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs is at or near the cleavage site in the antisense strand.

[0281] For dsRNAi agents having a duplex region 19-23 nucleotides in length, the cleavage site of the antisense strand is typically near positions 10, 11, 12 of the 5' end. Thus, the three identically modified motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide at the 5' end of the antisense strand or counting from the first paired nucleotide in the duplex region at the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary according to the length of the duplex region of the dsRNAi agent from the 5' end.

[0282] The sense strand of dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the break site of the strand; antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the break site of the strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned such that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

[0283] In some embodiments, the sense strand of the dsRNAi agent may contain more than one motif with three identical modifications on three consecutive nucleotides. The first motif may be at or near the cleavage site of the strand, and the other motif may be a wing modification. The term "wing modification" herein refers to a motif that is present in another part of the strand, separated from the motif at or near the cleavage site of the same strand. The wing modification is adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical properties of the motifs are significantly different from each other, and when the motifs are separated by one or more nucleotides, the chemical properties may be the same or different. There may be two or more wing modifications. For example, when there are two wing modifications, each wing modification may be present at one end relative to the first motif that is at or near the cleavage site, or on either side of the lead motif.

[0284] Similar to sense strand, antisense strand of dsRNAi agent may contain more than one motif with three identical modifications on three consecutive nucleotides, at least one of which is present at or near the break site of strand.This antisense strand may also contain one or more wing modifications in alignment, similar to the wing modifications that may be present on sense strand.

[0285] In some embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0286] In other embodiments, wing modifications on the sense or antisense strand of a dsRNAi agent typically do not include the first one or two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0287] When the sense and antisense strands of a dsRNAi agent each contain at least one wing modification, the wing modifications can be located at the same end of the duplex region and can have an overlap of 1, 2, or 3 nucleotides.

[0288] When the sense and antisense strands of a dsRNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that: two of each modification from one strand are located at one end of the duplex region and have an overlap of one, two, or three nucleotides; two of each modification from one strand are located at the other end of the duplex region and have an overlap of one, two, or three nucleotides; two of each modification from one strand are located on each side of the lead motif and have an overlap of one, two, or three nucleotides within the duplex region.

[0289] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNAi agent, including the nucleotide that is part of the motif, can be modified.Each nucleotide can be modified by the same or different modifications, which can include one or more of the modification of one or both of non-linked phosphate oxygens or one or more of linked phosphate oxygens; modification of the components of ribose sugar, for example, the 2'-hydroxyl on ribose sugar; replacement of phosphate moiety with "dephosphorylation" linker altogether; modification or replacement of naturally occurring base; and replacement or modification of ribose-phosphate backbone.

[0290] Nucleic acids are polymers of subunits, so many of the modifications, such as modifications of bases, or phosphate moieties, or non-linked O of phosphate moieties, occur at positions that are repeated in nucleic acids. In some cases, modifications will occur at all of the target positions in nucleic acids, but in many cases, they will not. By way of example, modifications may occur only at the 3'-end position or the 5'-end position, and may occur only in terminal regions, such as at positions on terminal nucleotides, or at the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in double-stranded regions of RNA, or only in single-stranded regions of RNA. For example, phosphorothioate modifications at non-linked O positions may occur only at one or both termini, only in the terminal regions, e.g., at the terminal nucleotide positions or in the last 2, 3, 4, 5, or 10 nucleotides of the strand, and in double-stranded and single-stranded regions, particularly at the termini. The 5' terminus or both termini may be phosphorylated.

[0291] For example, it may be possible to include specific bases in the overhang or to include modified nucleotides or nucleotide surrogates in the single-stranded overhang, for example, in the 5'-overhang or the 3'-overhang, or both, to enhance stability. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3'-overhang or the 5'-overhang may be modified, for example, by the modifications described herein. Modifications may include, for example, the use of modifications at the 2' position of the ribose sugar, for example, the use of 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modified deoxyribonucleotides rather than ribosugars in the nucleobases, and modifications in the phosphate group, for example, phosphorothioate modifications, with modifications known in the art. The overhang need not be homologous to the target sequence.

[0292] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. A strand may contain more than one modification. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0293] At least two different modifications are typically present on the sense and antisense strands, and these two modifications may be 2'-O-methyl, 2'-fluoro, or other modifications.

[0294] In certain embodiments, N a or N b includes modifications in an alternating pattern. As used herein, the term "alternating motif" refers to a motif having one or more modifications, with each modification occurring on alternating nucleotides of a strand. Alternating nucleotides may refer to one every two nucleotides, or one every three nucleotides, or a similar pattern. For example, where A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0295] The types of modifications contained within the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0296] In some embodiments, the dsRNAi agent of the present invention comprises a modification pattern for alternating motifs on the sense strand that is shifted compared to the modification pattern for alternating motifs on the antisense strand. The shift can be such that the modified nucleotides of the sense strand correspond to different modified nucleotides of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from the 5' to the 3' of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from the 5' to the 3' of the strand, in the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from the 5' to the 3' of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from the 5' to the 3' of the strand, in the duplex region, such that there is a complete or partial shift of the modification pattern between the sense strand and the antisense strand.

[0297] In some embodiments, dsRNAi agent comprises the alternating motif pattern of 2'-O-methyl modification and 2'-F modification on the initial sense strand, which has shift compared with the alternating motif pattern of 2'-O-methyl modification and 2'-F modification on the initial antisense strand, i.e., the 2'-O-modified nucleotide on the sense strand base pairs with the 2'-F modified nucleotide on the antisense strand, and vice versa.The 1st position of the sense strand may start with 2'-F modification, and the 1st position of the antisense strand may start with 2'-O-methyl modification.

[0298] The introduction of one or more motifs with three identical modifications on three consecutive nucleotides into sense strand or antisense strand interrupts the initial modification pattern existing in sense strand or antisense strand.The interruption of the modification pattern of sense strand or antisense strand by the introduction of one or more motifs with three identical modifications on three consecutive nucleotides into sense strand or antisense strand can enhance the gene silencing activity for target gene.

[0299] In some embodiments, when a motif of three identical modifications on three consecutive nucleotides is introduced into either of the strands, the modifications of the nucleotides adjacent to the motif are different modifications from the modification of the motif. For example, a portion of a sequence containing a motif may be a YYYN b ..." [In the sequence, "Y" represents a modification of the motif with three identical modifications on three consecutive nucleotides, and "N a " and "N b " represents a modification to the nucleotide adjacent to the motif "YYY" that is different from the modification of Y, in this case N a and N b may be the same or different modifications. Alternatively, if wing modifications are present, N a or N b may or may not be present.

[0300] The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur at any nucleotide at any position of the strand in the sense strand, the antisense strand, or both strands. For example, the internucleotide linkage modification may occur at every nucleotide on the sense strand or the antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or the antisense strand; the sense strand or the antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may have a shift compared to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at the 5' end or the 3' end.

[0301] In some embodiments, the dsRNAi agent comprises a modification with phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage in the protruding region.For example, the protruding region can contain two nucleotides with phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage between the two nucleotides.The modification with internucleotide linkage can also be made to link the protruding nucleotide with the paired nucleotide at the end in the double-stranded region.For example, at least two, three, four or all of the protruding nucleotides can be linked via phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage, but there can be an additional phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage that links the protruding nucleotide with the paired nucleotide adjacent to the protruding nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the three nucleotides at the end, where two of the three nucleotides are protruding nucleotides and the third nucleotide is the paired nucleotide adjacent to the protruding nucleotide. These terminal three nucleotides may be at the 3'-end of the antisense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, or the 5'-end of the antisense strand.

[0302] In some embodiments, the 2-nucleotide overhang is at the 3'-end of antisense strand, and there are two phosphorothioate internucleotide linkages between the three nucleotides at the end, where two of the three nucleotides are overhanging nucleotides, and the third nucleotide is the paired nucleotide adjacent to the overhanging nucleotide.In addition, the dsRNAi agent can have two phosphorothioate internucleotide linkages between the three nucleotides at the end at both the 5'-end of sense strand and the 5'-end of antisense strand.

[0303] In one embodiment, the dsRNAi agent contains mismatch(es) with the target in the duplex, or a combination thereof. The mismatch may occur in the overhang region or may occur in the duplex region. Base pairs may be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of association or dissociation of a particular pairing, the simplest approach is to consider the pairing at each base pair, but next neighbor or similar analysis may also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; pairings that include universal bases are preferred over canonical pairings.

[0304] In certain embodiments, the dsRNAi agent comprises at least one of the first one, two, three, four, or five base pairs in the duplex region at the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairs, or pairings that include a universal base, so as to promote dissociation of the antisense strand at the 5' end of the duplex.

[0305] In certain embodiments, the nucleotide at position 1 in the double-stranded region at the 5' end of antisense strand is selected from A, dA, dU, U and dT.Alternatively, at least one of the first one, two or three base pairs in the double-stranded region at the 5' end of antisense strand is AU base pair.For example, the first base pair in the double-stranded region at the 5' end of antisense strand is AU base pair.

[0306] In other embodiments, the nucleotide at the 3' end of the sense strand is deoxythymidine (dT) or the nucleotide at the 3' end of the antisense strand is deoxythymidine (dT). For example, there is a short sequence of deoxythymidine nucleotides, for example, two dT nucleotides, at the 3' end of the sense strand, the antisense strand, or both strands.

[0307] In certain embodiments, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3'(I) [In formula: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a represents oligonucleotide sequences that independently contain 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b represents, independently, an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and each n q independently represent an overhanging nucleotide; where Nb and Y do not have the same modification; XXX, YYY, and ZZZ each independently represent a motif with three identical modifications on three consecutive nucleotides. In one embodiment, YYY are all 2'-F modified nucleotides. It can be represented by:

[0308] In some embodiments, N a or N b includes modifications with alternating patterns.

[0309] In some embodiments, YYY motif is present at or near the cleavage site of sense strand.For example, when dsRNAi agent has a double-stranded region of 17-23 nucleotides in length, YYY motif can be present at or near the cleavage site (e.g., can be present at 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13) of sense strand, counting from the first nucleotide of 5' end; or counting in the first paired nucleotide in the double-stranded region of 5' end.

[0310] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib); 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) It can be represented by:

[0311] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0312] When the sense strand is represented by formula (Ic), Nb represents an oligonucleotide sequence that contains 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence that contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0313] When the sense strand is represented as formula (Id), each N b represents an oligonucleotide sequence that independently contains 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a can independently represent an oligonucleotide sequence that contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0314] Each of X, Y, and Z may be the same as or different from each other.

[0315] In other embodiments, i is 0, j is 0 and the sense strand has the formula: 5'n p -N a -YYY-N a -n q 3'(Ia) It can be represented by:

[0316] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence that contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0317] In one embodiment, the antisense strand sequence of the RNAi has the formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -np '3'(II) [In formula: k and l are each independently 0 or 1; p' and q' are each independently 0 to 6; each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differentially modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and each n q ' independently represents an overhanging nucleotide; Here, N b ' and Y' do not have the same modification; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif with three identical modifications on three consecutive nucleotides. It can be represented by:

[0318] In some embodiments, N a ' or N b ' includes modifications with alternating patterns.

[0319] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand.For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the Y'Y'Y' motif can be present at the 9th, 10th, 11th; 10th, 11th, 12th; 11th, 12th, 13th; 12th, 13th, 14th; or 13th, 14th, 15th positions of the antisense strand, counting from the first nucleotide of the 5' end; or counting at the first paired nucleotide in the duplex region of the 5' end.In one embodiment, the Y'Y'Y' motif is present at the 11th, 12th, 13th positions.

[0320] In certain embodiments, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0321] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0322] Thus, the antisense strand has the following formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb); 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc); or 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3'(IId) It can be represented by:

[0323] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' represents an oligonucleotide sequence that independently contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0324] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' represents an oligonucleotide sequence that independently contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0325] When the antisense strand is represented by formula (IId), each N b Each N' represents an oligonucleotide sequence that contains, independently, 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a In one embodiment, N' independently represents an oligonucleotide sequence that contains 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6.

[0326] In other embodiments, k is 0, l is 0 and the antisense strand has the formula: 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia) It can be represented by:

[0327] When the antisense strand is represented by formula (IIa), each N a X', Y', and Z' independently represent an oligonucleotide sequence containing from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides. Each of X', Y', and Z' can be the same or different from one another.

[0328] Each nucleotide of the sense strand and the antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' can specifically represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0329] In some embodiments, the sense strand of the dsRNAi agent may contain a YYY motif present at the 9th, 10th, and 11th positions of the strand, which may be counted from the first nucleotide at the 5'-end or counted at the first paired nucleotide in the 5'-end duplex region when the duplex region is 21nt; Y represents a 2'-F modification.The sense strand may additionally contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0330] In some embodiments, the antisense strand may contain a Y'Y'Y' motif present at positions 11, 12, 13 of the strand, which may be counted from the first nucleotide at the 5' end or counted at the first paired nucleotide within the duplex region at the 5' end; Y' represents a 2'-O-methyl modification. The antisense strand may additionally contain a X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe or 2'-F modification.

[0331] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0332] Thus, a dsRNAi agent for use in the methods of the invention can include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the iRNA duplex can have the formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -nq 3' Antisense:3'n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q '5' (III) [In formula: i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently 0 to 6; each N a and each N a ' independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differentially modified nucleotides; each N b and each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; where each of them may or may not be present, p ',n p , n q ', and n q independently represent an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif with three identical modifications on three consecutive nucleotides. It can be represented by:

[0333] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both of i and j are 0; or both of i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both of k and l are 0; or both of k and l are 1.

[0334] An exemplary combination of a sense strand and an antisense strand to form an iRNA duplex has the following formula: 5'n p -N a -YYY-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N a 'n q '5' (IIIa) 5'n p -N a -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-Y'Y'Y'-N b '-Z'Z'Z'-N a 'n q '5' (IIIb) 5'n p -N a -XXX-N b -YYY-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N a '-n q '5' (IIIc) 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3'n p '-N a '-X'X'X'-N b '-Y'Y'Y'-N b '-Z'Z'Z'-N a -n q '5' (IIId) Includes.

[0335] When the dsRNAi agent is represented by formula (IIIa), each N a represents an oligonucleotide sequence that independently contains from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0336] When the dsRNAi agent is represented by formula (IIIb), each N b represents an oligonucleotide sequence that independently contains 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a represents an oligonucleotide sequence that contains, independently, from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0337] When the dsRNAi agent is represented as formula (IIIc), each N b , N b Each N' represents an oligonucleotide sequence that contains, independently, 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a represents an oligonucleotide sequence that contains, independently, from 2 to 20, from 2 to 15, or from 2 to 10 modified nucleotides.

[0338] When the dsRNAi agent is represented as formula (IIId), each N b , N b Each N' represents an oligonucleotide sequence that contains, independently, 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a N' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b、 and N b Each of ' independently includes modifications with an alternating pattern.

[0339] Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same as or different from one another.

[0340] When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can form a base pair with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides form a base pair with the corresponding Y' nucleotide; or all three of the Y nucleotides form a base pair with the corresponding Y' nucleotide.

[0341] When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can form a base pair with one of the Z' nucleotides.Alternatively, at least two of the Z nucleotides form a base pair with the corresponding Z' nucleotide; or all three of the Z nucleotides form a base pair with the corresponding Z' nucleotide.

[0342] When the dsRNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides can form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides form a base pair with the corresponding X' nucleotide; or all three of the X nucleotides form a base pair with the corresponding X' nucleotide.

[0343] In certain embodiments, the modification on a Y nucleotide is different from the modification on a Y' nucleotide, the modification on a Z nucleotide is different from the modification on a Z' nucleotide, or the modification on an X nucleotide is different from the modification on an X' nucleotide.

[0344] In certain embodiments, when the dsRNAi agent is represented by formula (IIId), N aThe modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives joined via a bivalent or trivalent branched linker (described below). a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives that are joined via a divalent or trivalent branched linker.

[0345] In some embodiments, when the dsRNAi agent is represented by formula (IIIa), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives that are joined via a divalent or trivalent branched linker.

[0346] In some embodiments, dsRNAi agent is a multimer that contains at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), where the double strands are connected by a linker.The linker can be cleavable or non-cleavable.The multimer can further comprise a ligand.Each of the double strands can target the same gene, or can target two different genes; each of the double strands can target the same gene at two different target sites.

[0347] In some embodiments, dsRNAi agent is a multimer that contains 3, 4, 5, 6 or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), where double strands are connected by linker.Linker can be cleavable or non-cleavable.Multimer can further comprise ligand.Each of double strands can target the same gene or can target two different genes; each of double strands can target the same gene at two different target sites.

[0348] In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) can be linked with each other at one or both of 5'-end and 3'-end, and can be conjugated with ligand.Each agent can target the same gene, or can target two different genes; each agent can target the same gene at two different target sites.

[0349] In certain embodiments, the RNAi agent of the present invention may contain a small number of nucleotides containing 2'-fluoro modifications, for example, 10 or less nucleotides containing 2'-fluoro modifications. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2'-fluoro modifications. In a specific embodiment, the RNAi agent of the present invention contains 10 nucleotides with 2'-fluoro modifications, for example, 4 nucleotides with 2'-fluoro modifications in the sense strand and 6 nucleotides with 2'-fluoro modifications in the antisense strand. In another specific embodiment, the RNAi agent of the present invention contains 6 nucleotides with 2'-fluoro modifications in the sense strand, for example, 4 nucleotides with 2'-fluoro modifications and 2 nucleotides with 2'-fluoro modifications in the antisense strand.

[0350] In other embodiments, the RNAi agent of the present invention may contain only a few nucleotides that contain 2'-fluoro modification, for example, no more than two nucleotides that contain 2'-fluoro modification.For example, the RNAi agent may contain two, one, or zero nucleotides with 2'-fluoro modification.In a specific embodiment, the RNAi agent may contain two nucleotides with 2'-fluoro modification, for example, zero nucleotides with 2-fluoro modification in the sense strand, and two nucleotides with 2'-fluoro modification in the antisense strand.

[0351] A variety of publications describe multimeric iRNAs that can be used in the methods of the present invention, including WO2007 / 091269, U.S. Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0352] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of the RNAi agents described herein. In an exemplary embodiment, the 5' vinyl phosphonate modified nucleotides of the present disclosure have the structure:

[0353] [ka] [Wherein, X is O or S; R is hydrogen, hydroxy, fluoro or C 1~20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH) 2 and the C5' carbon and R 5’ the double bond between is in the E or Z orientation (e.g., the E configuration); and B is a nucleobase or a modified nucleobase, where B may be adenine, guanine, cytosine, thymine, or uracil. has.

[0354] The vinyl phosphonate of the present disclosure may be attached to the antisense strand or the sense strand of the dsRNA of the present disclosure.In certain embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA, and may be attached at the 5' end of the antisense strand of the dsRNA.

[0355] Vinyl phosphonate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphonate structures include those described above, where R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration [e.g., E configuration].

[0356] As described in more detail below, an iRNA containing one or more carbohydrate moieties conjugated to the iRNA may optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety will be conjugated to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA may be replaced by a non-carbohydrate (e.g., cyclic) carrier to which another moiety, e.g., a carbohydrate ligand, is attached. Herein, a ribonucleotide subunit in which the ribose sugar of the subunit is replaced in this manner is referred to as a ribose-replacement modified subunit (RRMS). The cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms are heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system or may contain two or more rings, e.g., fused rings. The cyclic carrier may be a fully saturated ring system or may contain one or more double bonds.

[0357] The ligand may be attached to the polynucleotide via the carrier. The carrier comprises (i) at least one "backbone junction", e.g., two "backbone junctions", and (ii) at least one "tethering junction". As used herein, "backbone junction" refers to a functional group, e.g., a hydroxyl group, or generally a bond that is available and suitable for incorporation of the carrier into the backbone of a ribonucleic acid, e.g., the phosphate backbone, or a modified phosphate backbone, e.g., a sulfur-containing backbone. In some embodiments, a "tethering junction" (TAP) refers to a ring atom, e.g., a carbon atom or a heteroatom (an atom significantly different from the atom that provides the backbone junction), that is a component of the cyclic carrier to which the selected moiety is attached. The moiety may be, for example, a carbohydrate, e.g., a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide. The selected moiety may be attached by interposing a tether to the cyclic carrier. Thus, the cyclic carrier will often contain a functional group, e.g., an amino group, or generally provide a bond suitable for incorporation or tethering of another chemical entity, e.g., a ligand, to the constituent ring.

[0358] The iRNA may be conjugated to the ligand via a carrier, which may be a cyclic or acyclic group; in one embodiment, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalin; in one embodiment, the acyclic group is a serinol backbone or a diethanolamine backbone.

[0359] a. Thermal instability modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermolabile modifications into the seed region of the antisense strand.As used herein, "seed region" refers to the 2-9 positions of the 5' end of the reference strand.For example, thermolabile modifications can be incorporated into the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

[0360] The term "thermolabile modification(s)" includes modification(s) that result in a dsRNA having an overall melting temperature (Tm) that is lower than the Tm of a dsRNA without such modification(s). For example, a thermolabile modification(s) can reduce the Tm of a dsRNA by 1-4°C, such as 1, 2, 3 or 4 degrees Celsius. Additionally, the term "thermolabile nucleotide" refers to a nucleotide that contains one or more thermolabile modifications.

[0361] It has been found that dsRNAs with antisense strands containing at least one duplex thermolabile modification within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one duplex (e.g., one, two, three, four, five, or more) thermolabile modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the one or more thermolabile modification(s) of the duplex are located within 2-9 positions, e.g., 4-8 positions, from the 5' end of the antisense strand. In some further embodiments, the duplex thermolabile modification(s) are located at 6, 7, or 8 positions from the 5' end of the antisense strand. In some still further embodiments, the duplex thermolabile modification is located at 7 positions of the 5' end of the antisense strand. In some embodiments, the thermolabile modifications of the duplex are located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0362] An iRNA agent includes a sense strand and an antisense strand, each strand having 14-40 nucleotides.

[0363] [ka] [In formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a nucleotide containing a modification selected from the group consisting of 2'-O-alkyl, 2'-substituted alkoxy, 2'-substituted alkyl, 2'-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide [2'-O-NMA, 2'O-CH2C(O)N(Me)H] modification] It can be represented by:

[0364] C1 is a thermolabile nucleotide located opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is at a position in the sense strand that pairs with a nucleotide at positions 2-8 of the 5' end of the antisense strand. In one example, C1 is at position 15 of the 5' end of the sense strand. The C1 nucleotide possesses a thermolabile modification that may include an abasic modification; a mismatch with the opposing nucleotide in the duplex; and a sugar modification such as a 2'-deoxy modification, or an acyclic nucleotide, e.g., an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In one embodiment, C1 is i) a mismatch with the opposing nucleotide in the antisense strand; ii)

[0365] [ka] and iii) an abasic modification selected from the group consisting of:

[0366] [ka]

[0023] wherein B is a modified or unmodified nucleobase; R 1 and R 2 are independently H, halogen, OR 3 or alkyl; R 3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermolabile modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; at least one nucleobase in the mismatch pair may be a 2'-deoxynucleobase. In one example, the thermolabile modification in C1 is GNA or

[0367] [ka] It is. T1, T1', T2', and T3 each independently represent a nucleotide that includes a modification that provides the nucleotide with a steric bulk equal to or less than that of the 2'-OMe modification. Steric bulk refers to the sum of the steric effects of the modifications. Those skilled in the art know methods for determining the steric effect of a modification of a nucleotide. The modification may be at the 2' position of the ribose sugar of the nucleotide, or may be a non-ribose nucleotide, an acyclic nucleotide, or a modification to the backbone of the nucleotide that is similar or equivalent to the modification at the 2' position of the ribose sugar, providing the nucleotide with a steric bulk equal to or less than that of the 2'-OMe modification. For example, T1, T1', T2', and T3' each independently are selected from DNA, RNA, LNA, 2'-F, and 2'-F-5'-methyl. In one embodiment, T1 is DNA. In one embodiment, T1' is DNA, RNA, or LNA. In one embodiment, T2' is DNA or RNA. In one embodiment, T3' is DNA or RNA. n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length. n 5 , q 3 , and q 7 are independently 1 to 6 nucleotide(s) in length. n 4 , q 2 , and q 6 are independently 1 to 3 nucleotide(s) in length; alternatively, n 4 is 0. q 5 is independently 0 to 10 nucleotide(s) in length. n 2 and q 4 are independently 0 to 3 nucleotide(s) in length.

[0368] Alternatively, n 4 is 0 to 3 nucleotides in length.

[0369] In one embodiment, n4 can be 0. In one example, n 4 is 0, and q 2 and q 6 is 1. In another example, n 4 is 0, and q 2 and q 6 is 1 with two phosphorothioate internucleotide linkages modified in positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), two phosphorothioate internucleotide linkages modified in positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and two phosphorothioate internucleotide linkages modified in positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0370] In one embodiment, n 4 , q 2 , and q 6 are each equal to 1.

[0371] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each equal to 1.

[0372] In one embodiment, C1 is at positions 14-17 of the 5' end of the sense strand when the sense strand is 19-22 nucleotides in length; 4 is 1. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0373] In one embodiment, T3' begins at position 2 of the 5' end of the antisense strand. 6 is equal to 1.

[0374] In one embodiment, T1' begins at position 14 of the 5' end of the antisense strand. 2 is equal to 1.

[0375] In an exemplary embodiment, T3' begins at position 2 of the 5' end of the antisense strand, and T1' begins at position 14 of the 5' end of the antisense strand. In one example, T3' begins at position 2 of the 5' end of the antisense strand, and q 6 is equal to 1, T1' starts from position 14 of the 5' end of the antisense strand, and q 2 is equal to 1.

[0376] In one embodiment, T1' and T3' are separated by a length of 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0377] In one embodiment, T1' is at position 14 of the 5' end of the antisense strand. 2 is equal to 1, and modifications at the 2' position, or at non-ribose, acyclic or backbone positions, provide less steric bulk than 2'-OMe ribose.

[0378] In one embodiment, T3' is at position 2 of the 5' end of the antisense strand. 6 is equal to 1, and modifications at the 2' position, or at non-ribose, acyclic or backbone positions, result in less steric bulk than 2'-OMe ribose.

[0379] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is at position 11 of the 5' end of the sense strand when the sense strand is 19-22 nucleotides in length; 2 is 1. In an exemplary embodiment, T1 is at the cleavage site of the sense strand, at position 11 of the 5' end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n 2 is 1.

[0380] In one embodiment, T2' begins at position 6 of the 5' end of the antisense strand. In one example, T2' is at positions 6-10 of the 5' end of the antisense strand, and 4 is 1.

[0381] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., at position 11 of the 5' end of the sense strand, when the sense strand is 19 to 22 nucleotides in length, 2 is 1; T1' is at position 14 of the 5' end of the antisense strand; q 2 is equal to 1, the modification to T1' is at the 2' position of the ribose sugar or at a non-ribose, acyclic or backbone position that is less sterically bulky than 2'-OMe ribose; T2' is at the 6-10 position of the 5' end of the antisense strand, and q 4 is 1; T3' is at position 2 of the 5' end of the antisense strand; q 6 is equal to 1 and the modifications to T3' are at the 2' position or at non-ribose, acyclic or backbone positions that provide steric bulk less than 2'-OMe ribose.

[0382] In one embodiment, T2' begins at position 8 of the 5' end of the antisense strand. 4 is 2.

[0383] In one embodiment, T2' begins at position 9 of the 5' end of the antisense strand. 4 is 1.

[0384] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0385] In one embodiment, n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0386] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0387] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with modification with two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0388] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 6, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0389] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 6, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0390] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0391] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0392] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and optionally with at least two additional TTs at the 3' end of the antisense strand.

[0393] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; optionally with at least two additional TTs at the 3'-end of the antisense strand; with modification with two phosphorothioate internucleotide linkages within positions 1 to 5 of the sense strand (counting from the 5'-end of the sense strand), with modification with two phosphorothioate internucleotide linkages within positions 1 and 2 of the antisense strand (counting from the 5'-end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages within positions 18 to 23 of the antisense strand (counting from the 5'-end of the antisense strand).

[0394] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0395] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages in positions 1 to 5 of the sense strand (counting from the 5' end), with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages in positions 18 to 23 of the antisense strand (counting from the 5' end).

[0396] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0397] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with modification with two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0398] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0399] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages at positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with modification with two phosphorothioate internucleotide linkages at positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

[0400] The RNAi agent may contain a phosphorus-containing group at the 5' end of the sense or antisense strand. The phosphorus-containing group at the 5' end may be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS 2 ), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy-5'-C-malonyl (

[0401] [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5'-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., a trans-vinyl phosphonate,

[0402] [ka] ), 5'-Z-VP isomers (i.e., cis-vinyl phosphonates,

[0403] [ka] ), or a mixture thereof.

[0404] In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5'-end of the antisense strand.

[0405] In one embodiment, the RNAi agent comprises a 5'-P. In one embodiment, the RNAi agent comprises a 5'-P in the antisense strand.

[0406] In one embodiment, the RNAi agent comprises a 5'-PS. In one embodiment, the RNAi agent comprises a 5'-PS in the antisense strand.

[0407] In one embodiment, the RNAi agent comprises a 5'-VP. In one embodiment, the RNAi agent comprises a 5'-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5'-E-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5'-Z-VP in the antisense strand.

[0408] In one embodiment, the RNAi agent is a 5'-PS 2 In one embodiment, the RNAi agent comprises in the antisense strand a 5'-PS 2 Includes.

[0409] In one embodiment, the RNAi agent is a 5'-PS 2 In one embodiment, the RNAi agent comprises in the antisense strand, 5'-deoxy-5'-C-malonyl.

[0410] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0411] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0412] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0413] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is also a 5'-PS 2 Also includes.

[0414] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0415] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0416] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

[0417] In one embodiment, B1 is 2'-OMe or 2'-F, 1is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0418] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-PS 2 Also includes.

[0419] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0420] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0421] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0422] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0423] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 The RNAi agent is also a 5'-PS 2 Also includes.

[0424] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0425] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-P.

[0426] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-PS.

[0427] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with a modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with a modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0428] In one embodiment, B1 is 2'-OMe or 2'-F,1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5'-PS 2 Also includes.

[0429] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0430] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0431] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0432] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0433] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. dsRNAi RNA (dsRNAi RNA) also contains 5'-PS 2 Also includes.

[0434] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0435] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0436] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

[0437] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0438] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-PS 2 Also includes.

[0439] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0440] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-P.

[0441] In one embodiment, B1 is 2'-OMe or 2'-F, 1is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also contains a 5'-PS.

[0442] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0443] In one embodiment, B1 is 2'-OMe or 2'-F,1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 The RNAi agent is also a 5'-PS 2 Also includes.

[0444] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. RNAi agents also include 5'-deoxy-5'-C-malonyl.

[0445] In one embodiment, B1 is 2'-OMe or 2'-F,1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P.

[0446] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS.

[0447] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0448] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-PS 2 Also includes.

[0449] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl.

[0450] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0451] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), with modification with two phosphorothioate internucleotide linkages. The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0452] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof), and a targeting ligand.

[0453] In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0454] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-PS 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0455] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0456] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end) of the antisense strand, and with modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0457] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end) of the antisense strand, and with modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0458] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with a modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end) of the antisense strand, and with a modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof) and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0459] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and with modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5'-PS 2and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0460] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages in positions 1-5 (counting from the 5' end) of the sense strand, with a modification with two phosphorothioate internucleotide linkages in positions 1 and 2 (counting from the 5' end) of the antisense strand, and with a modification with two phosphorothioate internucleotide linkages in positions 18-23 (counting from the 5' end) of the antisense strand. The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0461] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0462] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0463] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof), and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0464] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-PS 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0465] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0466] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-P and a targeting ligand. In one embodiment, the 5'-P is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0467] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-PS and a targeting ligand. In one embodiment, the 5'-PS is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0468] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a combination thereof), and a targeting ligand. In one embodiment, the 5'-VP is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0469] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5'-PS 2 and a targeting ligand. 2 is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0470] In one embodiment, B1 is 2'-OMe or 2'-F, 1 is 8, T1 is 2'F, n 2 is 3, B2 is 2'-OMe, n 3 is 7, and n 4 is 0, B3 is 2'-OMe, n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; with a modification with two phosphorothioate internucleotide linkages at positions 1-5 of the sense strand (counting from the 5' end of the sense strand), with a modification with two phosphorothioate internucleotide linkages at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand), and with a modification with two phosphorothioate internucleotide linkages at positions 18-23 of the antisense strand (counting from the 5' end of the antisense strand). The RNAi agent also includes 5'-deoxy-5'-C-malonyl and a targeting ligand. In one embodiment, the 5'-deoxy-5'-C-malonyl is at the 5' end of the antisense strand and the targeting ligand is at the 3' end of the sense strand.

[0471] In certain embodiments, the RNAi agent of the invention comprises (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; and (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14-16, 18, and 20 (counting from the 5' end). and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23, and 2'F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end) and an antisense strand having Includes; In this case, the dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0472] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0473] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-6, 8, 10, and 12-21, 2'-F modifications at positions 7 and 9, and a deoxynucleotide (e.g., dT) at position 11 (counting from the 5' end); and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23, and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0474] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2'-F modifications at positions 7, 9, 11, 13, and 15; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0475] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-9, and 12-21, and 2'-F modifications at positions 10, and 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23, and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0476] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14-21; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23, and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0477] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 10, 12, 14, 15, 17, and 19-21, and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 25 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23, 2'-F modifications at positions 2, 3, 5, 8, 10, 12, 14, 16, and 18, and deoxynucleotides (e.g., dT) at positions 24 and 25 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a four nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0478] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0479] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 21 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2'-F modifications at positions 7, and 9 to 11; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 23 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0480] In another particular embodiment, the RNAi agent of the invention comprises: (a) a sense strand, (i) 19 nucleotides in length; (ii) an ASGPR ligand conjugated to the 3' end, the ASGPR ligand comprising three GalNAc derivatives conjugated via a trivalent branched linker; (iii) 2'-OMe modifications at positions 1-4, 6, and 10-19, and 2'-F modifications at positions 5, and 7-9; and (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5' end) and a sense strand having (b) an antisense strand, (i) 21 nucleotides in length; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5' end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end). and an antisense strand having Includes; In this case, the RNAi agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0481] In certain embodiments, the iRNA for use in the methods of the invention is an agent selected from those listed in any one of Tables 4-5. These agents may further comprise a ligand.

[0482] v. Antisense polynucleotide agent containing a motif In certain embodiments of the present invention, at least one of the consecutive nucleotides of the antisense polynucleotide agent of the present invention may be a modified nucleotide. In one embodiment, the modified nucleotide comprises one or more modified sugars. In another embodiment, the modified nucleotide comprises one or more modified nucleobases. In yet another embodiment, the modified nucleotide comprises one or more modified internucleoside linkages. In some embodiments, the modifications (sugar modifications, nucleobase modifications, or linkage modifications) define a pattern or motif. In one embodiment, the patterns of modifications of the sugar moiety, the internucleoside linkage, and the nucleobase are each independent of each other.

[0483] Antisense polynucleotide agents with modified oligonucleotides arranged in a pattern or motif can provide drug properties such as enhanced inhibitory activity, increased binding affinity to target nucleic acid, or resistance to in vivo nuclease degradation.For example, such agents can contain at least one region that is modified to provide increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to target nucleic acid, or increased inhibitory activity.The second region of such agents can be used as a substrate for the intracellular endonuclease RNaseH, which cleaves the RNA strand of an RNA:DNA duplex.

[0484] An exemplary antisense polynucleotide agent having modified oligonucleotides arranged in a pattern or motif is a gapmer.In a "gapmer", an internal region or "gap" having a number of linked nucleotides that support RNaseH cleavage is located between two external linked regions or "wings" having a number of linked nucleotides that are chemically different from the linked nucleotides of the internal region.The gap segment is usually used as a substrate for endonuclease cleavage, but the wing segments contain modified nucleotides.

[0485] The three regions of a gapmer motif (5'-wing, gap, and 3'-wing) form a contiguous sequence of nucleotides and may be described as "XYZ", where "X" represents the length of the 5-wing, "Y" represents the length of the gap, and "Z" represents the length of the 3'-wing. In one embodiment, a gapmer described as "XYZ" has a structure in which a gap segment is located immediately adjacent to each of the 5'-wing and 3'-wing segments. Thus, there are no intervening nucleotides between the 5'-wing segment and the gap segment, or between the gap segment and the 3'-wing segment. Any of the antisense compounds described herein may have a gapmer motif. In some embodiments, X and Z are the same, and in other embodiments, they are different.

[0486] In certain embodiments, the regions of a gapmer are differentiated by the type of modified nucleotides within the region. In some embodiments, the types of modified nucleotides that can be used to differentiate the regions of a gapmer include β-D-ribonucleotides, β-D-deoxyribonucleotides, 2'-modified nucleotides, such as 2'-modified nucleotides (e.g., 2'-MOE, and 2'-O-CH3), and bicyclic sugar modified nucleotides (e.g., those having a 4'-(CH2)nO-2' bridge, where n=1 or n=2).

[0487] In one embodiment, at least some of the modified nucleotides of each wing may be different from at least some of the modified nucleotides of the gap. For example, at least some of the modified nucleotides of each wing closest to the gap (the 3'-most nucleotide of the 5'-wing and the 5'-most nucleotide of the 3-wing) are different from the modified nucleotides of the nearby gap nucleotides, thus defining a boundary between the wing and the gap. In certain embodiments, the modified nucleotides within a gap are identical to each other. In certain embodiments, the gap contains one or more modified nucleotides that are different from the modified nucleotides of one or more other nucleotides of the gap.

[0488] The length of the 5'-wing of a gapmer (X) can be from 1 to 6 nucleotides in length, e.g., from 2 to 6, 2 to 5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to 4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0489] The length of the 3'-wing of a gapmer (Z) can be from 1 to 6 nucleotides in length, e.g., from 2 to 6, 2 to 5, 3 to 6, 3 to 5, 1 to 5, 1 to 4, 1 to 3, 2 to 4 nucleotides in length, e.g., 1, 2, 3, 4, 5, or 6 nucleotides in length.

[0490] The length of the gap (Y) of a gapmer may be 5-14 nucleotides in length, e.g., 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-14, 8-13, 8-12 , 8-11, 8-10, 8-9, 9-14, 9-13, 9-12, 9-11, 9-10, 10-14, 10-13, 10-12, 10-11, 11-14, 11-13, 11-12, 12-14, 12-13, or 13-14 nucleotides in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.

[0491] In some embodiments of the invention, X consists of 2, 3, 4, 5, or 6 nucleotides, Y consists of 7, 8, 9, 10, 11, or 12 nucleotides, and Z consists of 2, 3, 4, 5, or 6 nucleotides.Such gapmers include (XYZ)2-7-2, 2-7-3, 2-7-4, 2-7-5, 2-7-6, 3-7-2, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7-6, 3-7-3, 3-7-4, 3-7-5, 3-7-6, 4-7-3, 4-7-4, 4-7-5, 4-7-6, 5-7-3, 5-7-4, 5-7-5, 5-7-6, 6-7-3, 6-7-4, 6-7-5, 6-7- 6, 2-8-2, 2-8-3, 2-8-4, 2-8-5, 2-8-6, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-8-6, 4-8-3, 4-8-4, 4-8-5, 4-8-6, 5-8-3, 5-8-4, 5-8-5, 5-8-6, 6-8-3, 6-8-4, 6-8-5, 6-8-6, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-9-6, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-6, 4-9-3, 4-9-4, 4-9-5, 4-9-6, 5-9-3, 5-9-4, 5-9-5, 5-9-6, 6-9 -3, 6-9-4, 6-9-5, 6-9-6, 2-10-2, 2-10-3, 2-10-4, 2-10-5, 2-10-6, 3-10-2, 3-10-3, 3-10-4, 3-10-5, 3-10-6, 4-10-3, 4-10-4, 4-10-5, 4-10-6, 5-10-3, 5-10-4, 5-10-5, 5-10-6, 6-10-3, 6-10-4, 6-10-5, 6-10-6, 2-11-2, 2-11-3, 2-11-4, 2-11-5, 2-11-6, 3-11-2, 3-11-3, 3-11-4, 3-11-5, 3-11-6 , 4-11-3, 4-11-4, 4-11-5, 4-11-6, 5-11-3, 5-11-4, 5-11-5, 5-11-6, 6-11-3, 6-11-4, 6-11-5, 6-11-6, 2-12-2, 2-12-3, 2-12-4, 2-12-5, 2-12-6, 3-12-2, 3-12-3, 3-12-4, 3-12-5, 3-12-6, 4-12-3, 4-12-4, 4-12-5, 4-12-6, 5-12-3, 5-12-4, 5-12-5, 5-12-6, 6-12-3, 6-12-4, 6-12-5, or 6-12-6.

[0492] In some embodiments of the invention, an antisense polynucleotide agent targeting INHBE comprises a 5-10-5 gapmer motif. In other embodiments of the invention, an antisense polynucleotide agent targeting INHBE comprises a 4-10-4 gapmer motif. In another embodiment of the invention, an antisense polynucleotide agent targeting INHBE comprises a 3-10-3 gapmer motif. In yet other embodiments of the invention, an antisense polynucleotide agent targeting INHBE comprises a 2-10-2 gapmer motif.

[0493] The 5'-wing or 3'-wing of a gapmer can, independently, contain from 1 to 6 modified nucleotides, for example, 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0494] In some embodiments, the 5'-wing of the gapmer comprises at least one modified nucleotide. In one embodiment, the 5'-wing of the gapmer comprises at least two modified nucleotides. In another embodiment, the 5'-wing of the gapmer comprises at least three modified nucleotides. In yet another embodiment, the 5'-wing of the gapmer comprises at least four modified nucleotides. In another embodiment, the 5'-wing of the gapmer comprises at least five modified nucleotides. In certain embodiments, each nucleotide of the 5'-wing of the gapmer is a modified nucleotide.

[0495] In some embodiments, the 3'-wing of the gapmer comprises at least one modified nucleotide. In one embodiment, the 3'-wing of the gapmer comprises at least two modified nucleotides. In another embodiment, the 3'-wing of the gapmer comprises at least three modified nucleotides. In yet another embodiment, the 3'-wing of the gapmer comprises at least four modified nucleotides. In another embodiment, the 3'-wing of the gapmer comprises at least five modified nucleotides. In certain embodiments, each nucleotide of the 3'-wing of the gapmer is a modified nucleotide.

[0496] In certain embodiments, the regions of the gapmer are differentiated by the type of sugar moiety of the nucleotides. In one embodiment, the nucleotides of each different region contain the same sugar moiety. In other embodiments, the nucleotides of each different region contain different sugar moieties. In certain embodiments, the sugar nucleotide modification motifs of the two wings are the same as each other. In certain embodiments, the sugar nucleotide modification motif of the 5'-wing is different from the sugar nucleotide modification motif of the 3'-wing.

[0497] The 5'-wing of a gapmer can contain from 1 to 6 modified nucleotides, for example 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0498] In one embodiment, at least one modified nucleotide in the 5'-wing of the gapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide, or LNA. In another embodiment, the 5'-wing of the gapmer comprises 2, 3, 4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide in the 5'-wing of the gapmer is a bicyclic nucleotide.

[0499] In one embodiment, the 5'-wing of the gapmer comprises at least 1, 2, 3, 4, or 5 constrained ethyl nucleotides. In some embodiments, every nucleotide of the 5'-wing of the gapmer is a constrained ethyl nucleotide.

[0500] In one embodiment, the 5'-wing of the gapmer comprises at least one LNA nucleotide. In another embodiment, the 5'-wing of the gapmer comprises 2, 3, 4, or 5 LNA nucleotides. In other embodiments, every nucleotide in the 5'-wing of the gapmer is an LNA nucleotide.

[0501] In certain embodiments, at least one modified nucleotide in the 5'-wing of a gapmer is a non-bicyclic modified nucleotide, e.g., a 2'-substituted nucleotide. A "2'-substituted nucleotide" is a nucleotide that contains a modification at the 2' position that is other than H or OH, e.g., a 2'-OMe nucleotide, or a 2'-MOE nucleotide. In one embodiment, the 5'-wing of a gapmer contains 2, 3, 4, or 5 2'-substituted nucleotides. In one embodiment, each nucleotide in the 5'-wing of a gapmer is a 2'-substituted nucleotide.

[0502] In one embodiment, the 5'-wing of the gapmer comprises at least one 2'-OMe nucleotide. In one embodiment, the 5'-wing of the gapmer comprises at least 2, 3, 4, or 5 2'-OMe nucleotides. In one embodiment, each nucleotide of the 5'-wing of the gapmer comprises a 2'-OMe nucleotide.

[0503] In one embodiment, the 5'-wing of the gapmer comprises at least one 2'-MOE nucleotide. In one embodiment, the 5'-wing of the gapmer comprises at least 2, 3, 4, or 5 2'-MOE nucleotides. In one embodiment, each nucleotide of the 5'-wing of the gapmer comprises a 2'-MOE nucleotide.

[0504] In certain embodiments, the 5'-wing of the gapmer comprises at least one 2'-deoxynucleotide. In certain embodiments, each nucleotide of the 5'-wing of the gapmer is a 2'-deoxynucleotide. In certain embodiments, the 5'-wing of the gapmer comprises at least one ribonucleotide. In certain embodiments, each nucleotide of the 5'-wing of the gapmer is a ribonucleotide.

[0505] The 3'-wing of a gapmer can contain from 1 to 6 modified nucleotides, for example, 1, 2, 3, 4, 5, or 6 modified nucleotides.

[0506] In one embodiment, at least one modified nucleotide in the 3'-wing of the gapmer is a bicyclic nucleotide, such as a constrained ethyl nucleotide, or LNA. In another embodiment, the 3'-wing of the gapmer comprises 2, 3, 4, or 5 bicyclic nucleotides. In some embodiments, each nucleotide in the 3'-wing of the gapmer is a bicyclic nucleotide.

[0507] In one embodiment, the 3'-wing of the gapmer comprises at least one constrained ethyl nucleotide. In another embodiment, the 3'-wing of the gapmer comprises 2, 3, 4, or 5 constrained ethyl nucleotides. In some embodiments, each nucleotide of the 3'-wing of the gapmer is a constrained ethyl nucleotide.

[0508] In one embodiment, the 3'-wing of the gapmer comprises at least one LNA nucleotide. In another embodiment, the 3'-wing of the gapmer comprises 2, 3, 4, or 5 LNA nucleotides. In other embodiments, every nucleotide of the 3'-wing of the gapmer is an LNA nucleotide.

[0509] In certain embodiments, at least one modified nucleotide in the 3'-wing of the gapmer is a non-bicyclic modified nucleotide, e.g., a 2'-substituted nucleotide. In one embodiment, the 3'-wing of the gapmer comprises 2, 3, 4, or 5 2'-substituted nucleotides. In one embodiment, each nucleotide in the 3'-wing of the gapmer is a 2'-substituted nucleotide.

[0510] In one embodiment, the 3'-wing of the gapmer comprises at least one 2'-OMe nucleotide. In one embodiment, the 3'-wing of the gapmer comprises at least 2, 3, 4, or 5 2'-OMe nucleotides. In one embodiment, each nucleotide of the 3'-wing of the gapmer comprises a 2'-OMe nucleotide.

[0511] In one embodiment, the 3'-wing of the gapmer comprises at least one 2'-MOE nucleotide. In one embodiment, the 3'-wing of the gapmer comprises at least 2, 3, 4, or 5 2'-MOE nucleotides. In one embodiment, each nucleotide of the 3'-wing of the gapmer comprises a 2'-MOE nucleotide.

[0512] In certain embodiments, the 3'-wing of the gapmer comprises at least one 2'-deoxynucleotide. In certain embodiments, each nucleotide of the 3'-wing of the gapmer is a 2'-deoxynucleotide. In certain embodiments, the 3'-wing of the gapmer comprises at least one ribonucleotide. In certain embodiments, each nucleotide of the 3'-wing of the gapmer is a ribonucleotide.

[0513] The gap of a gapmer may contain from 5 to 14 modified nucleotides, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 modified nucleotides.

[0514] In one embodiment, the gapmer gap contains at least one 5-methylcytosine. In one embodiment, the gapmer gap contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 5-methylcytosines. In one embodiment, all of the nucleotides of the gapmer gap are 5-methylcytosines.

[0515] In one embodiment, the gapmer gap comprises at least one 2'-deoxynucleotide. In one embodiment, the gapmer gap comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 2'-deoxynucleotides. In one embodiment, all of the nucleotides of the gapmer gap are 2'-deoxynucleotides.

[0516] Gapmers may contain one or more modified internucleotide linkages. In some embodiments, gapmers contain one or more phosphodiester internucleotide linkages. In other embodiments, gapmers contain one or more phosphorothioate internucleotide linkages.

[0517] In one embodiment, each nucleotide of the 5'-wing of the gapmer is linked via a phosphorothioate internucleotide linkage. In another embodiment, each nucleotide of the 3'-wing of the gapmer is linked via a phosphorothioate internucleotide linkage. In yet another embodiment, each nucleotide of the gap segment of the gapmer is linked via a phosphorothioate internucleotide linkage. In one embodiment, all of the nucleotides in the gapmer are linked via phosphorothioate internucleotide linkages.

[0518] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and positioned between a 5'-wing segment comprising 5 nucleotides and a 3'-wing segment comprising 5 nucleotides.

[0519] In another embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and positioned between a 5'-wing segment comprising 4 nucleotides and a 3'-wing segment comprising 4 nucleotides.

[0520] In another embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides positioned immediately adjacent to and between a 5'-wing segment comprising 3 nucleotides and a 3'-wing segment comprising 3 nucleotides.

[0521] In another embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and positioned between a 5'-wing segment comprising 2 nucleotides and a 3'-wing segment comprising 2 nucleotides.

[0522] In one embodiment, each nucleotide of the 5-wing adjacent to a gap segment of ten 2'-deoxyribonucleotides comprises a modified nucleotide. In another embodiment, each nucleotide of the 3-wing adjacent to a gap segment of ten 2'-deoxyribonucleotides comprises a modified nucleotide. In one embodiment, each modified 5'-wing nucleotide and each modified 3'-wing nucleotide comprises a 2'-sugar modification. In one embodiment, the 2'-sugar modification is a 2'-OMe modification. In another embodiment, the 2'-sugar modification is a 2'-MOE modification. In one embodiment, each modified 5'-wing nucleotide and each modified 3'-wing nucleotide comprises a bicyclic nucleotide. In one embodiment, the bicyclic nucleotide is a constrained ethyl nucleotide. In another embodiment, the bicyclic nucleotide is an LNA nucleotide. In one embodiment, each cytosine in an antisense polynucleotide agent targeting an INHBE gene is a 5-methylcytosine.

[0523] In one embodiment, the antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and located between a 5'-wing segment comprising 5 nucleotides comprising 2'OMe modifications and a 3'-wing segment comprising 5 nucleotides comprising 2'OMe modifications, and each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine. In some embodiments, the agent further comprises a ligand.

[0524] In one embodiment, the antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and located between a 5'-wing segment comprising 5 nucleotides comprising a 2'MOE modification and a 3'-wing segment comprising 5 nucleotides comprising a 2'MOE modification, and each internucleotide linkage of the agent is a phosphorothioate linkage. In one embodiment, each cytosine of the agent is a 5-methylcytosine. In some embodiments, the agent further comprises a ligand.

[0525] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and positioned between a 5'-wing segment comprising five constrained ethyl nucleotides and a 3'-wing segment comprising five constrained ethyl nucleotides, and each internucleotide linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0526] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and located between a 5'-wing segment comprising five LNA nucleotides and a 3'-wing segment comprising five LNA nucleotides, and each internucleotide linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0527] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and positioned between a 5'-wing segment comprising 4 nucleotides comprising 2'OMe modifications and a 3'-wing segment comprising 4 nucleotides comprising 2'OMe modifications, and each internucleotide linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0528] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and positioned between a 5'-wing segment comprising 4 nucleotides comprising a 2'MOE modification and a 3'-wing segment comprising 4 nucleotides comprising a 2'MOE modification, and each internucleotide linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0529] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and positioned between a 5'-wing segment comprising four constrained ethyl nucleotides and a 3'-wing segment comprising four constrained ethyl nucleotides, and each internucleotide linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0530] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleotides immediately adjacent to and located between a 5'-wing segment comprising four LNA nucleotides and a 3'-wing segment comprising four LNA nucleotides, and each internucleotide linkage of the agent is a phosphorothioate linkage. In some embodiments, each cytosine of the agent is a 5-methylcytosine.

[0531] In one embodiment, an antisense polynucleotide agent targeting the INHBE gene comprises a gap segment of 10 2'-deoxyribonucleo...

Claims

1. Modulators of inhibin subunit beta E (INHBE).

2. 2. The modulator of claim 1, wherein the modulator is selected from the group consisting of an oligonucleotide that targets an INHBE, an antibody or antigen-binding fragment thereof that specifically binds to an INHBE, a small molecule, a guide RNA that performs ADAR editing, and a guide RNA that performs CRISPR editing.

3. 3. The modulator of claim 2, wherein the oligonucleotide targeting INHBE is a double-stranded ribonucleic acid (dsRNA) or an antisense polynucleotide agent.

4. The modulator of claim 2 , wherein the antibody or antigen-binding fragment thereof that specifically binds to INHBE is a human monoclonal anti-INHBE antibody or antigen-binding fragment thereof.

5. The modulator of claim 2 , wherein the guide RNA comprises a stem-loop structure that binds to the ADAR enzyme.

6. The dsRNA comprises a sense strand and an antisense strand that form a double-stranded region, and the nucleotide sequences of the sense strand and the antisense strand are as shown in the following table: 【Table 1】 【Table 2】 【Table 3】 【Table 4】 【Table 5】 【Table 6】 【Table 7】 【Table 8】 4. The modulator of claim 3, comprising any one of the sense and antisense strand nucleotide sequences provided in

7. 4. The modulator of claim 3, wherein the antisense polynucleotide agent comprises 4 to 50 consecutive nucleotides, at least one of the consecutive nucleotides is a modified nucleotide, and the nucleotide sequence of the agent is 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 1, 2, 4, 6, 8, or 10.

8. The antisense polynucleotide agent is selected from the group consisting of: 【Table 9】 【Table 10】 【Table 11】 【Table 12】 4. The modulator of claim 3, comprising at least 8 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the nucleotide sequences listed in 9. The modulator of claim 3, wherein all of the nucleotides of the dsRNA or antisense polynucleotide agent comprise nucleotide modifications.

10. The modulator of claim 3, wherein the sense strand and antisense strand of the dsRNA are each independently 18 to 30 nucleotides in length, or the antisense polynucleotide agent is 18 to 30 nucleotides in length.

11. The modulator of claim 9, wherein the nucleotide modification comprises a sugar moiety modification selected from the group consisting of a 2'-O-methoxyethyl sugar moiety modification, a 2'-methoxy sugar moiety modification, a 2'-O-alkyl sugar moiety modification, and a bicyclic sugar moiety modification.

12. The modulator described in claim 9, wherein the nucleotide modification is a 5-methylcytosine modification.

13. A modulator as described in claim 9, wherein the nucleotide modification includes an internucleoside linkage modification.

14. The modulator described in claim 11, comprising multiple 2'-deoxynucleotides flanked on both sides by at least one nucleotide having a sugar moiety modification.

15. The antisense polynucleotide agent is a gap segment consisting of linked deoxynucleotides; a 5'-wing segment consisting of linked nucleotides; a 3'-wing segment consisting of linked nucleotides wherein a gap segment is positioned between the 5'-wing segment and the 3'-wing segment, and each nucleotide of each wing segment comprises a sugar moiety modification.

16. 16. The modulator of claim 15, wherein the gap segment is 10 2'-deoxynucleotides in length and each wing segment is 5 nucleotides in length.

17. The modulator described in claim 15, wherein the sugar moiety modification is selected from the group consisting of a 2'-O-methoxyethyl sugar moiety modification, a 2'-methoxy sugar moiety modification, a 2'-O-alkyl sugar moiety modification, and a bicyclic sugar moiety modification.

18. 16. The modulator of claim 15, wherein all nucleotides comprise modified internucleoside linkages.

19. The modulator of claim 3 further comprising a ligand.

20. 20. The modulator of claim 19, which is conjugated to a ligand at the 3' end.

21. 20. The modulator of claim 19, wherein the ligand is an N-acetylgalactosamine (GalNac) derivative.

22. The ligand is 【Chemical 1】 22. The modulator of claim 21, wherein:

23. A pharmaceutical composition for inhibiting the expression and / or activity of INHBE, comprising a modulator according to any one of claims 1-22.

24. The pharmaceutical composition of claim 23, further comprising a lipid formulation.

25. 1. An in vitro method of inhibiting the expression and / or activity of INHBE in a cell, comprising: (a) contacting a cell with a modulator according to any one of claims 1-22; and (b) maintaining the cells produced in step (a) for a time sufficient to obtain inhibition of INHBE expression and / or activity, thereby inhibiting the expression and / or activity of INHBE in the cells. An in vitro method comprising:

26. 26. The method of claim 25, wherein INHBE expression and / or activity is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or to a level below the level of detection.

27. 24. The pharmaceutical composition of claim 23 for treating or preventing at least one symptom in a subject having a disorder that would benefit from reduced inhibin subunit beta E (INHBE) expression and / or activity.

28. 28. The pharmaceutical composition of claim 27, wherein the disorder is an INHBE-associated disorder.

29. 29. The pharmaceutical composition of claim 28, wherein the INHBE-related disorder is selected from the group consisting of metabolic disorders, cardiovascular diseases, and hypertension.

30. 30. The pharmaceutical composition of claim 29, wherein the metabolic disorder is metabolic syndrome.

31. 28. The pharmaceutical composition of claim 27, wherein the subject is a human.

32. 28. The pharmaceutical composition of claim 27, wherein the modulator is administered subcutaneously to the subject.

33. 28. The pharmaceutical composition of claim 27, wherein an additional therapeutic agent for the treatment of an INHBE-associated disorder is further administered to the subject.

34. A kit, vial, or syringe comprising a modulator according to any one of claims 1-22.

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