Advanced RNA targeting (arnatar) for inhbe

Abstract

Disclosed herein are Advanced RNA Targeting (ARNATAR) dsRNA compounds targeting Inhibin βE (INHBE). Such compounds are useful in methods for reducing expression of INHBE and for therapeutically treating INHBE associated diseases, disorders and / or conditions, or symptoms thereof in a subject.

Description

[0001] ADVANCED RNA TARGETING (ARNATAR) FOR INHBE

[0002] FIELD

[0003] Certain embodiments are directed to methods and compounds for modulating Inhibin PE (INHBE) gene expression by Advanced RNA Targeting (ARNATAR). Such methods and compounds are usefu l for reducing expression of INHBE, thereby treating diseases, disorders and / or conditions related to INHBE in a subject.

[0004] BACKGROUND

[0005] The use of therapeutic oligomeric compounds (e.g., oligonucleotides) was first proposed over forty years ago by Stephenson and Zamecnik (Inhibition of Rous Sarcoma Viral RNA Translation by a Specific Oligodeoxyribonucleotide, PNAS, 1978, 75:285-288).

[0006] Sequence-specific silencing of gene expression, RNA interference (RNAi), was discovered in 1998 by Fire et al. (Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans, Nature, 1998, 391:806-811). RNAi utilizes double-stranded RNA (dsRNA) to inhibit gene expression via the RNA-induced silencing complex (RISC).

[0007] RISC comprises a complex of multiple proteins interacting with an oligomeric compound to inhibit gene expression. The oligomeric compound acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcripts to target a specific mRNA transcript for cleavage. Cleavage of the target mRNA blocks translation of the target mRNA and silences the target gene. Oligomeric compounds utilized by RISC include, but are not limited to: single stranded oligomeric compounds such as microRNAs (miRNAs), certain oligonucleotides, and single strand siRNAs (Lima et al., Single-stranded siRNAs activate RNAi in animals. Cell. 2012, 150(5):883-94), and double-stranded RNA (dsRNA) compounds such as short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs).

[0008] In 2001, Elbashir et al. showed that 21-nucleotide long siRNA duplexes specifically suppress expression of endogenous and heterologous genes in mammalian cell lines and theorized that siRNA may eventually be used as a gene-specific therapeutic (Duplexes of 21 -Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells, Nature, 2001 , 411 :494-498).

[0009] Inhibin PE is also known as Inhibin Subunit Beta E, INHBE or Activin E. In 2018, Sugiyama et al. identified INHBE as a hepatokine with hepatic expression that correlated positively with insulin resistance and body mass index (BMI) in humans. Additionally, INHBE expression was increased in the livers of a rodent model of Type II Diabetes. (Sugiyama et al, PL-oS ONE 13(3): e0194798. hitps: / / doi .org / 10.1371 / iournal. pone.0194798). Cao el al. showed that INHBE was up-regulated in a steatosis hepatocyte model and in animals fed a high fat diet. Cao et al. identified INHBE to be a hub gene for non-alcoholic fatty liver disease (NAFLD). (Cao el al. , Biochem Biophys Res Common., 2023 Dec 17:686: 149180). Accordingly, reducing INHBE expression in liver has the potential to treat INHBE related diseases, disorder and / or conditions including, but not limited to, metabolic, cardiovascular, hypertensive, inflammatory, cancerous, neurodegenerative, and / or hepatosteatotic diseases, disorders and / or conditions.

[0010] Disclosed herein are dsRNA compounds targeting INHBE improved with Advanced RNA Targeting (ARNATAR) abilities that enhance their gene silencing activity for use in reducing INHBE expression and treating INHBE related diseases, disorders and / or conditions in a subject.

[0011] SUMMARY

[0012] Several embodiments provided herein relate to the discovery of certain ARNATAR design dsRNA compounds targeting INHBE transcripts that can enhance their effectiveness in modulating INHBE gene expression. In several aspects, the dsRNA compound is a modified shRNA or siRNA compound. The double-stranded RNA compound comprises a sense strand and an antisense strand. The antisense strands can be fully or substantially complementary to a target nucleic acid.

[0013] In some embodiments, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell comprises a sense strand and an antisense strand forming tire dsRNA compound, wherein the antisense strand comprises or is any one of the antisense strand sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In some embodiments, the antisense strand is in a salt form.

[0014] In some embodiments, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the sense strand comprises or is any one of the sense strand sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In some embodiments, the sense strand is in a salt form. In some embodiments, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the sense strand comprises or is any one of the sense strand sequences in any one of Tables 2, 6, 8, 10, 12 or 14, and wherein the antisense strand comprises or is the corresponding antisense strand sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In some embodiments, the sense strand and / or the antisense strand is in a salt form.

[0015] In certain embodiments, the dsRNA compound comprises a conjugate. In certain embodiments, the conjugate is an N-Acetylgalactosamine-compri sing moiety (GalNAc). In certain embodiments, the conjugate is attached to the 3* end of the sense strand of a dsRNA compound targeting INHBE. In certain embodiments, the conjugate is attached to the 3* end of the sense strand of a siRNA compound targeting INHBE.

[0016] Certain embodiments provide a method for inhibiting the expression of INHBE in a subject comprising the step of administering a dsRNA compound described herein to the subject, in an amount sufficient to inhibit expression of INHBE. Tn certain embodiments, the dsRNA compound inhibits expression of INHBE by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In certain embodiments, the dsRNA compound comprises a siRNA.

[0017] In certain embodiments, a pharmaceutical composition for inhibiting expression of INHBE in a cell is provided comprising a double-stranded ribonucleic acid (dsRNA) compound described herein, alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0018] Certain embodiments provide a method of inhibiting expression of INHBE in a cell comprising contacting the cell with a dsRNA compound or a pharmaceutical composition described herein in an amount sufficient to inhibit expression of INHBE, thereby inhibiting expression of INHBE in the cell.

[0019] Certain embodiments provide a method of treating and / or preventing an INHBE associated disease, disorder and / or condition in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a dsRNA compound or a pharmaceutical composition described herein, thereby treating and / or preventing the INHBE associated disease, disorder and / or condition in the subject.

[0020] Certain embodiments provide a method of treating a subject having a disease, disorder and / or condition that would benefit from reduction in INHBE expression, comprising administering to the subject in need thereof a therapeutically effective amount of a dsRNA compound or a pharmaceutical composition described herein, thereby treating the subject having the disease, disorder and-'or condition that would benefit from reduction in INHBE expression.

[0021] Certain embodiments provide a method of preventing at least one symptom in a subject having a disease, disorder and / or condition that would benefit from reduction in INHBE expression, comprising administering to the subject in need thereof a prophylactically effective amount of a dsRNA compound or a pharmaceutical composition described herein, thereby preventing at least one symptom in the subject having the disease, disorder and / or condition that would benefit from reduction in INHBE expression.

[0022] In certain embodiments, a kit comprising a dsRNA compound or a pharmaceutical composition described herein, and optionally, a label, is provided.

[0023] In one embodiment, a process for preparing a sense and / or antisense strand of a double- stranded ribonucleic acid (dsRNA) compound described herein is provided, wherein the process comprises the steps of: a) preparing the sense and / or antisense strand by sequential coupling of modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis on a solid support; b) optionally, coupling an N-Acetylgalactosamine-comprising moiety (GalNAc) to the sense and / or antisense strand on the solid support via the phosphoramidite oligonucleotide synthesis; c) detaching the sense and / or antisense strand from the solid support and removing the solid support; and d) optionally, further purifying the sense and / or antisense strand, optionally using chromatography.

[0024] In one embodiment, a process for preparing a sense and / or antisense strand of a double- stranded ribonucleic acid (dsRNA) compound described herein is provided, wherein the process comprises the steps of: a) coupling an N-Acetylgalactosamine-comprising moiety (GalNAc) to a solid support via the phosphoramidite oligonucleotide synthesis, b) coupling a modified and / or unmodified nucleotide via the phosphoramidite oligonucleotide synthesis to the GalNAc on the solid support; c) sequentially coupling additional modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis to prepare the sense and / or anisense strand; d) detaching the sense and / or antisense strand from the solid support and removing the solid support; and e) optionally, further purifying the sense and / or antisense strand, optionally using chromatography.

[0025] In one embodiment, a process of preparing a double-stranded ribonucleic acid (dsRNA) compound described herein is provided, comprising: a) contacting the sense strand prepared according to any one of the processes described herein with the antisense strand prepared according to any one of the processes described herein in equimolar concentrations in a solution; b) optionally healing (he solution lo a temperature of about 94°C; and c) optionally, reducing the temperature of the solution lo about 25°C.

[0026] Certain embodiments provide a double stranded-ribonucleic acid (dsRNA) compound described herein for use in medicine.

[0027] Certain embodiments provide a double-stranded ribonucleic acid (dsRNA) compound described herein for use in treating or preventing an INHBE associated disease, disorder and / or condition in a subject.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGURE 1: shows INHBE siRNA inhibition of INHBE mRNA in Hep3B cells 18 hours after transfection. INHBE siRNAs ATsi962-ATsi968 were assessed with reference siRNA ATsi972.

[0030] FIGURE 2: shows INHBE siRNA inhibition of INHBE mRNA in human primary' hepatocyte cells 72 hours after free uptake. INHBE siRNAs ATsi963, ATsi965, ATsi967 were assessed with reference siRNA ATsi972.

[0031] FIGURE 3: shows INHBE siRNA inhibition of INHBE mRNA in Hep3B cells 16 hours after transfection. INHBE siRNAs ATsi967, ATsi974-ATsi979 were assessed with reference siRNA ATsi972.

[0032] FIGURE 4: shows INHBE siRNA inhibition of INHBE mRNA in Hep3B cells 16 hours after transfection. INHBE siRNAs ATsi967, ATsi978, ATsi979, ATsi980, ATsi981 , ATsi982, ATsi984 were assessed with reference siRNA ATsi972.

[0033] FIGURE 5: shows INHBE siRNA inhibition of INHBE mRNA in Hep3B cells 24 hours after transfection. INHBE siRNAs ATsi965, ATsi967, ATsi1O31-ATsi1O43 were assessed with reference siRNA ATsi972.

[0034] FIGURE 6: shows INHBE siRNA inhibition of INHBE mRNA in Hep3B cells 24 hours after transfection. INHBE siRNAs ATsi967, ATsi1032, ATsi1O36-ATsi1O38, ATsi1040, ATsi1O42, ATsi1169-ATsi1l74 were assessed with reference siRNA ATsi972.

[0035] FIGURE 7: shows Western Blots probed for INHBE protein levels in the liver of AAV infected BALB / c mice 4.5 weeks after administering INHBE siRNAs to the mice. FIGURE 8: shows a chart of INHBE protein levels in the liver of AAV infected BALB / c mice 4.5 weeks after administering INHBE siRNAs to the mice.

[0036] FIGURE 9: shows Western Blots probed for INHBE protein levels in the liver of AAV infected BALB / c mice 4.5 weeks after administering INHBE siRNAs to the mice.

[0037] FIGURE 10: shows a chart of INHBE protein levels in the liver of AAV infected BALB / c mice 4.5 weeks after administering INHBE siRNAs to the mice.

[0038] FIGURE 11: shows INHBE siRNA inhibition of INHBE mRNA in human primary hepatocyte cells 64 hours after free uptake. INHBE siRNAs ATsi980, ATsi1225, ATsi1173, ATsi1226, ATsi1227 were assessed with reference siRNA ATsi972.

[0039] FIGURE 12: shows INHBE siRNA inhibition of INHBE mRNA in Hep3B cells 24 hours after transfection. INHBE siRNAs ATsi1226, ATsi1227, ATsi1253, ATsi1254, ATsi1255, and ATsi1256 were assessed.

[0040] FIGURE 13: shows INHBE siRNA inhibition of INHBE mRNA in human primary hepatocyte cells 64 hours after free uptake. INHBE siRNAs ATsil 226, ATsil 227, ATsi1253, ATsi1254, ATsi1255, and ATsi1256 were assessed with reference siRNA ATsi972.

[0041] FIGURE 14: shows a) a protocol for treating AAV-hsINHBE BALB / c infected mice with INHBE siRNAs, and b) a graph of INHBE protein levels at days 10, 21, 35, and 57 in the liver of the AAV infected mice after administering INHBE siRNAs to the mice.

[0042] FIGURE 15: shows a) a protocol for treating AAV-hsINHBE BALB / c infected mice with IN HBE siRNAs, and b) a graph of INHBE protein levels at days 12, 22, 34, and 47 in the liver of the AAV infected mice after administering INHBE siRNAs to the mice.

[0043] DETAILED DESCRIPTION

[0044] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0045] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, GENBANK Accession Numbers, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

[0046] Definitions

[0047] Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated-by-reference for the portions of the document discussed herein, as well as in their entirety.

[0048] Unless otherwise indicated, the following terms have the following meanings:

[0049] “2’-O-methoxyethyl” (also 2’-M0E and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2’ position of a furanose ring. A 2"-O-methoxyethyl modified sugar is a modified sugar.

[0050] “2’-M0E nucleoside” (also 2’-O-methoxyethyl nucleoside) means a nucleoside comprising a 2’-M0E modified sugar moiety. “2’-M0E nucleotide” (also 2’-O-methoxyethyl nucleotide) means a nucleotide comprising a 2’-M0E modified sugar moiety.

[0051] “2’-O-methyl” (also 2’-OCH3 and 2’-0Me) refers to an O-methyl modification at the 2' position of a furanose ring. A 2’-O-methyl modified sugar is a modified sugar.

[0052] “2’-0Me nucleoside” (also 2’-O-methyl nucleoside) means a nucleoside comprising a 2’- OMe modified sugar moiety. “2’-0Me nucleotide” (also 2'-O-methyl nucleotide) means a nucleotide comprising a 2’-OMe modified sugar moiety.

[0053] “2’-substituted nucleoside” means a nucleoside comprising a substituent at the 2’-position of the furanose ring other than H or OH. In certain embodiments, 2’-substituted nucleosides include nucleosides with a fluoro (2’-F), O-methyl (2’-0Me), O-methoxyethyl (2 ’-MOE) or bicyclic sugar modifications.

[0054] “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

[0055] “About” means when used before a numerical designation (e.g., temperature, time, amount, concentration, and such other, including a range) indicates approximations which may vary by within ±7% of a value, e.g., 16%, 5%, 4%, 3%, 2%, or 1%. For example, if it is stated, “the compounds affected at least about 70% inhibition of mRNA”, it is implied that the mRNA levels are inhibited within a range of 63% and 77%. Recitation of ranges of values herein are merely intended to serve as a method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0056] “Animal” refers to a human or non-human animal, including, but not limited to, any of mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0057] “Antibody” refers to a complete antibody molecule or any fragment or portion thereof, such as any of the heavy chain, the light chain, Fabportion, and Fcportion.

[0058] “Antisense oligonucleotide” or “ASO” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid. In certain embodiments, the antisense oligonucleotide comprises one or more ribonucleoside (RNA) residues and / or deoxyribonucleoside (DNA) residues.

[0059] “Base complementarity” refers to the capacity for the base pairing of nucleobases of an oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and may be mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases. Base complementarity also refers to canonical (e.g., A:U, A:T, C:G) or non-canonical base pairings (e.g., A:G, A:U, G:U, 1:U, 1:A, I:C).

[0060] “Bicyclic sugar” means a compound wherein two rings, e.g., two furanose rings, are modified by the bridging of two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.

[0061] “Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an oligomeric compound. “Chemical modification” means modification of molecular structure or element from naturally occurring molecules. For example, siRNA compounds are composed of linked ribonucleosides (also sometimes referred to herein as RNA), therefore, substitution of a deoxyribonucleoside (also sometimes referred to herein as DNA) for a ribonucleoside is considered a chemical modification of the siRNA compound.

[0062] “Chemically distinct portion" refers to a portion of an oligomeric compound that is in some way chemically different than another portion of the same oligomeric compound. For example, a portion having 2’-OMe nucleotides is chemically distinct from a portion having nucleotides without 2’-0Me modifications.

[0063] “Chimeric oligomeric compounds” means oligomeric compounds that have at least 2 chemically distinct portions, each portion having a plurality of subunits. For example, as disclosed herein, siRNA can comprise a peripheral portion and a central portion. The peripheral portion comprises motifs with various modified or unmodified nucleobases so as to confer increased stability, specificity, safety and potency, while the central portion comprises various modified or unmodified nucleobases to serve as substrate for RISC mediated degradation.

[0064] “Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0065] “Comply” in the context of a therapy means the adherence with a recommended therapy by a subject.

[0066] “Comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated member without the exclusion of other (e.g., non-stated) members. For example, the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements, respectively. The terms “comprising," “having,” “including," and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

[0067] “Contiguous nucleobases” means nucleobases adjacent to each other.

[0068] “Deoxyribonucleotide" means a nucleotide having a hydrogen at the 2’ position of the sugar portion of the nucleotide. A sequence of deoxyribonucleotides is sometimes referred to as “DNA" herein. A deoxyribonucleotide is sometimes referred to as “DNA nucleotide”, “d nucleotide” or “D” herein. Deoxyribonucleotides may be modified with any of a variety of substituents. “Designing” or “designed” in the context of an oligomeric compound refers to the process of making / 'engineering an oligomeric compound that specifically hybridizes with a target nucleic acid molecule. Designing generally encompasses providing mutations and / or modifications to a e.g., natural sequence.

[0069] “Efficacy” means the ability to produce a desired effect.

[0070] “Expression” generally includes all the functions by which a gene’s coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and / or translation. Within the present disclosure, expression refers to protein as the products of expression.

[0071] “Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid molecule has a complementary nucleobase in a second nucleic acid molecule. In certain embodiments, a first nucleic acid molecule is an oligomeric compound and a target nucleic acid molecule is a second nucleic acid molecule.

[0072] “Fully modified motif’ refers to an oligomeric compound comprising a contiguous sequence of nucleosides wherein each nucleoside has a chemical modification.

[0073] “GalNAc” refers to the compound N-Acetylgalactosamine or to a compound comprising one or more N -Acetylgalactosamine compounds or moieties. An N-Acetylgalactosamine- comprising moiety describes a compound comprising at least one N-Acetylgalactosamine compound or moiety usually attached to one or more spacers and / or linkers for the attachment to an oligonucleotide compound.

[0074] “Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, a combination of an oligomeric compound and a nucleic acid molecule target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, a combination of a strand of an siRNA and a nucleic acid molecule target, particularly an mRNA target molecule.

[0075] “Induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease” or the like, generally denotes an action to obtain quantitative differences between two states.

[0076] “Inhibiting the expression or activity” refers to a reduction or blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity. “Intemucleoside linkage” refers to the chemical bond between two adjacent nucleosides. The linkage may be a naturally occurring linkage, i.e., a phosphate linkage, or an artificial linkage, such as a phosphorothioate (also known as thiophosphate or PS) linkage.

[0077] “Linked nucleosides” means adjacent nucleosides (e.g., A, G, C, T, U, and I) linked together by an intemucleoside linkage. Examples of linked nucleosides include a sequence of deoxyribonucleosides (sometimes referred to as DNA herein) or a sequence of ribonucleosides (sometimes referred to as RNA herein).

[0078] “Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid through Watson-Crick base-pairing (e.g., A:T, A:U, C:G).

[0079] “Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring intemucleoside bond (i.e., a phosphodiester intemucleoside bond).

[0080] “Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0081] “Modified nucleoside” means a nucleoside having a modified sugar moiety and / or a modified nucleobase. As used herein, where the oligomeric compound is RNA-based, a substitution of a deoxyribonucleoside (sometimes referred to as DNA nucleoside herein) for a ribonucleoside is considered a modification of the oligomeric compound. Also, where the oligomeric compound is DNA-based, a substitution of a ribonucleoside (sometimes referred to as RNA nucleoside herein) for a deoxyribonucleoside is considered a modification of the oligomeric compound.

[0082] “Modified nucleotide” means a nucleotide having at least one of a modified sugar moiety, a modified intemucleoside linkage, a deoxyribonucleoside (sometimes referred to as DNA nucleotide herein) for a ribonucleoside (sometimes referred to as RNA nucleotide herein) substitution, a ribonucleoside (sometimes referred to as RNA nucleoside herein) for a deoxyribonucleoside (sometimes referred to as DNA nucleoside herein) substitution, and a modified nucleobase.

[0083] “Modified oligonucleotide” means an oligonucleotide comprising at least one of a modified intemucleoside linkage, a modified sugar, a deoxyribonucleoside (sometimes referred to as DNA nucleoside herein) for a ribonucleoside (sometimes referred to as RNA nucleoside herein) substitution, a ribonucleoside (sometimes referred to as RNA nucleoside herein) for a deoxyribonucleoside (sometimes referred to as DNA nucleoside herein) substitution, and / or a modified nucleobase.

[0084] “Modified sugar” means substitution and / or any change from a natural sugar moiety of a nucleotide found in DNA or RNA.

[0085] “Moiety” means one of the portions into which something is divided i.e., a part or component of something. For example, a sugar moiety of a nucleotide is the sugar component of the nucleotide.

[0086] “Monomer” refers to a single unit of an oligomer or a single unit for forming an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

[0087] “Motif* means a pattern of modification in an oligomeric compound. For example, as disclosed herein, ARNATAR designed oligomeric compounds comprising motifs with various modified nucleobases and internucleoside linkages in order to improve delivery, stability, specificity, safety and potency of the compounds.

[0088] “Natural sugar (moiety)” or generally “sugar (moiety)” means a sugar (moiety) found in DNA (2'-H) or RNA (2' -OH), i.e., 2-deoxy-beta-D-ribofuranose or beta-D-ribofuranose, respectively.

[0089] “Naturally occurring intemucleoside linkage” means a 3’ to 5’ phosphodiester linkage.

[0090] “Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

[0091] “Nucleic acid (molecule)” refers to a sequence of monomeric nucleotides. A nucleic acid molecule includes, but is not limited to, ribonucleic acids (RNA), messenger RNA (mRNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), hairpin ribonucleic acids (shRNA), and microRNAs (miRNA).

[0092] “Nucleobase” means any unmodified nucleobase as defined above, any modified nucleobase and / or any artificial nucleobase that may generally be any heterocyclic moiety capable of pairing with a base of a nucleic acid molecule.

[0093] “Nucleobase complementarity” refers to the ability of a nucleobase of base pairing (also known as being complementary) with another nucleobase. If a nucleobase at a certain position of an oligomeric compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid molecule, then the position of hydrogen bonding between the oligomeric compound and the target nucleic acid molecule is considered to be complementary at that nucleobase pair. For example, in DNA, adenine (A) is complementary to thymine (T); in RNA, adenine (A) is complementary to uracil (U); and, guanine (G) is complementary to cytosine (C) in both DNA and RNA. Base pairs, or complementary nucleobases, are usually canonical Watson- Crick base pairs (C:G, A:U, A:T), but, non-canonical base pairs such as Hoogsteen base pairs (e.g., A:G, A:U), Wobble base pairs (e.g., G:U, I:U, I:A, I:C, wherein I is hypoxanthine) and the like are also included. Nucleobase complementarity facilitates hybridization of the oligomeric compounds described herein to their target nucleic acids.

[0094] “Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and / or nucleobase modification.

[0095] “Nucleoside” means a nucleobase linked to the natural or modified sugar as defined above. “Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetics include those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound, such as, for example peptide nucleic acids or morpholines (morpholinos linked by -N(H)-C(::::O)-O- or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and / or intemucleoside linkage. Often, a mimetic is used in place of the sugar or sugar- intemucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

[0096] “Nucleotide” means a nucleoside having a linkage group (e.g., a phosphate (p) or phosphorothioate (PS) group) covalently linked to the sugar portion of the nucleoside. Nucleotides include ribonucleotides and deoxyribonucleotides with phosphate and / or phosphorothioate linkages. A sequence of linked nucleotides, e.g., ribonucleotides, deoxyribonucleotides and / or a mixture thereof, form an oligonucleotide.

[0097] “Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of R.NA or protein expression of a gene other than the intended target nucleic acid.

[0098] “Oligomeric activity” means any detectable or measurable activity attributable to the hybridization of an oligomeric compound to its target nucleic acid. In certain embodiments, oligomeric activity is measured as a decrease in the amount or expression of a target nucleic acid. Oligomeric activity can be modulated by an oligomeric compound such as a dsRNA compound. Oligomeric activity can be modulated by an oligomeric compound such as an siRNA compound.

[0099] “Oligomeric compound” means a compound of linked monomeric subunits (also known as “subunits” herein) that is capable of undergoing hybridization to at least a region of a target nucleic acid through hydrogen bonding. The monomeric subunits are particularly modified or unmodified nucleotides or nucleosides, and the oligomeric compound is particularly an oligonucleotide. The oligomeric compound acts as a template for RISC to recognize complementary messenger RNA (mRNA) transcripts to target a specific mRNA transcript for cleavage. Cleavage of the target mRNA blocks translation of the target mRNA and silences the target gene. Examples of oligomeric compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, ssRNAs, siRNAs, shRNAs and miRNAs. Generally, oligomeric compounds are distinguished from polymeric compounds based on their number of monomers, wherein an oligomer often has about 5 to about 100 monomeric units.

[0100] “Oligomeric inhibition” means reduction of target nucleic acid (e.g., mRNA) levels in the presence of an oligomeric compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the oligomeric compound.

[0101] “Oligomeric mechanisms" include RISC or RNase II related mechanisms involving hybridization of an oligomeric compound with target nucleic acid (e.g., mRNA ), wherein the outcome or effect of the hybridization is target degradation and inhibition of gene expression.

[0102] “Oligonucleotide” as used herein means a sequence of linked nucleosides and / or nucleotides, each of which can be modified or unmodified, independently from each other. Oligonucleotides can have a linking group other than a phosphate group (e.g., a phosphorothioate group) used as a linking moiety between nucleosides. In certain embodiments, an oligonucleotide comprises one or more ribonucleoside (RNA) residues and / or deoxyribonucleoside (DNA) residues.

[0103] “Phosphorothioate linkage" or “PS" means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[0104] “Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a terget nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound.

[0105] “Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

[0106] “RNA” or “ribonucleic acid" consists of ribose nucleotides or ribonucleotides (nitrogenous bases attached to a ribose sugar) linked by phosphodiester bonds, forming sequences of varying lengths. The nitrogenous bases in natural RNA are adenine, guanine, cytosine, and uracil. In the dsRNA described herein, they may also comprise thymine and / or other bases.

[0107] “Ribonucleotide” means a nucleotide having a hydroxy at the 2’ position of the sugar portion of the nucleotide. Ribonucleotides can be modified with any of a variety of substituents and may be connected by covalent linkages other than naturally occurring phosphodiester such as phosphorothioate. A ribonucleotide is sometimes referred to as RNA, “R” or “r” herein.

[0108] “Segments” are defined as smaller or sub-portions of regions within a nucleic acid molecule, particularly a target nucleic acid.

[0109] “Sites" as used herein are defined as unique nucleobase positions within a nucleic acid, particularly a target nucleic acid.

[0110] “Specifically hybridizable" refers to an oligomeric compound having a sufficient degree of complementarity between an oligomeric compound (e.g., siRNA) and a target nucleic acid (e.g., mRNA) to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.

[0111] “Stringent hybridization conditions" or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target nucleic acid, with minimal hybridization to other nucleic acid molecules. “Subject" means a human or non-human animal, particularly a human or non-human animal selected for treatment or therapy.

[0112] “Target” generally refers to a protein or nucleic acid molecule, the modulation of which is desired. As used herein, “target” particularly refers to a nucleic acid molecule (e.g., mRNA), the modulation of which is desired.

[0113] “Target gene" refers to a gene encoding a target.

[0114] “Targeting” means the process of design and selection of an oligomeric compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

[0115] “Target nucleic acid," “target RNA,” “target RNA transcript,” and “nucleic acid target” all mean a nucleic acid capable of being targeted by oligomeric compounds.

[0116] “Target region" means a portion of a target nucleic acid to which one or more oligomeric compounds is targeted.

[0117] “Target segment” means the sequence of nucleotides of a target nucleic acid to which an oligomeric compound is targeted. “5’ target site" refers to the S’-most nucleotide of a target segment. “3' target site” refers to the 3 ’-most nucleotide of a target segment. In an embodiment, a target segment is at least a 13-nucleobase portion (i.e. at least 13 consecutive nucleobases) of a target region to which an oligomeric compound is targeted.

[0118] “Therapeutic efficacy” or “therapeutically effective" refers to the effectiveness of a compound or composition, such as an oligomeric compound described herein, in a therapeutic application. Therapeutic efficacy can be increased by improvements in delivery, stability, specificity, safety, and / or potency of the therapeutic compound.

[0119] “Unmodified” RNA nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). “Unmodified" DNA nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T) and cytosine (C). In certain embodiments, an unmodified RNA nucleobase is considered modified when a DNA nucleobase is substituted for the RNA nucleobase in an oligomeric compound such as an siRNA compound. In certain embodiments, an unmodified DNA nucleobase is considered modified when an RNA nucleobase is substituted for the DNA nucleobase in a DNA sequence.

[0120] “Unmodified nucleoside” means herein a nucleoside composed of a naturally occurring nucleobase and a naturally occurring sugar moiety. In certain embodiments, an unmodified nucleoside is an RNA nucleoside in an oligomeric compound such as an siRNA compound. “Unmodified nucleotide” means herein a nucleotide composed of a naturally occurring nucleobase, a naturally occurring sugar moiety, and an internucleoside linkage, wherein the intemucleotide linkage may be a naturally occurring linkage (i.e., a phosphate linkage) or an artificial linkage (e.g., a phosphorothioate linkage). In certain embodiments, an unmodified nucleotide is an RNA nucleotide in an oligomeric compound such as an siRNA compound.

[0121] Disclosed herein are improved compounds targeting Inhibin Beta E (INHBE) with Advanced RNA Targeting (ARNATAR) designs that enhance their gene silencing activity. In certain embodiments, the compound is a double-stranded ribonucleic acid (dsRNA) compound. In an embodiment, the dsRNA is an shRNA or an siRNA compound.

[0122] In one embodiment, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the antisense strand comprises or is any of the antisense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the dsRNA is an siRNA compound, wherein the antisense strand comprises or is any of the antisense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the siRNA comprises antisense nucleotide sequences as shown in Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the antisense nucleotide sequences shown in Tables 2, 6, 8, 10, 12 or 14 can be chemically modified with Advanced RNA Targeting (ARNATAR) motifs (reference is made to WO2024137543 which is incorporated-by-reference herein) in order to improve speed, stability, durability, specificity, safety and / or potency of siRNA compounds. In certain embodiments, the antisense nucleotide sequences correspond to the nucleotide sequences of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 151, 152, ora 13, 14, 15, 16, 17, 18, 19 or 20 nucleoside long portion thereof. In a preferred embodiment, the siRNA compound comprises the antisense nucleotide sequence of ATal223 (SEQ ID NO: 74), ATal224 (SEQ ID NO: 76), ATal253 (SEQ ID NO: 80), ATal254 (SEQ ID NO: 81), or a 13, 14, 15, 16, 17, 18, 19 or 20 nucleoside long portion thereof. In some embodiments, the antisense strand is in a salt form.

[0123] In one embodiment, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the sense strand comprises or is any of the sense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the dsRNA is an siRNA compound, wherein the sense strand comprises or is any of the sense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the siRNA comprises sense nucleotide sequences as shown in Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the sense nucleotide sequences shown in Tables 2, 6, 8, 10, 12 or 14 can be chemically modified with Advanced RNA Targeting (ARNATAR) motifs (reference is made to WO2024137543 which is incorporated-by-reference herein) in order to improve speed, stability, durability, specificity, safety and / or potency of siRNA compounds. In certain embodiments, the sense nucleotide sequences correspond to the nucleotide sequences of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 149, 150, or a 13, 14, 15, 16, 17, 18, 19 or 20 nucleoside long portion thereof. In a preferred embodiment, the siRNA compound comprises the sense nucleotide sequence of ATsl226 (SEQ ID NO: 75), ATS1227 (SEQ ID NO: 77), or a 13, 14, 15, 16, 17, 18, 19 or 20 nucleoside long portion thereof. In some embodiments, the sense strand is in a salt form.

[0124] In one embodiment a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of 1NHBE in a cell comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the sense strand comprises or is any of the sense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14, and wherein the antisense strand comprises or is any of the antisense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the dsRNA is an siRNA compound, wherein the sense strand comprises or is any of the sense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14, and wherein the antisense strand comprises or is any of the antisense nucleotide sequences in any one of Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the sense strand comprises a 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleoside long portion of any sense nucleotide sequence shown in Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the antisense strand comprises a 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleoside long portion of any antisense nucleotide sequence shown in Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the antisense nucleotide sequences correspond to the nucleotide sequence of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 103, 105, 107, 109, 111, 1 13, 115, 117, 1 19, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145. 147, 151 or 152, and the sense nucleotide sequences correspond to the nucleotide sequence of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 149 or 150. In a preferred embodiment, the sense strand comprises the nucleotide sequence of any of ATsl226 (SEQ ID NO: 75), ATsl227 (SEQ ID NO: 77), or a 13, 14, 15, 16, 17, 18, 19 or 20 nucleoside long portion thereof. In a preferred embodiment, tlie antisense strand comprises the nucleotide sequence of any of ATal223 (SEQ ID NO: 74), ATal224 (SEQ ID NO: 76), ATal253 (SEQ ID NO: 80), ATal254 (SEQ ID NO: 81), or a 13, 14, 15, 16, 17, 18, 19 or 20 nucleoside long portion thereof. In some embodiments, the sense and / or antisense strand is in a salt form.

[0125] In certain embodiments, the dsRNA compound comprises at least one modified nucleotide. In another embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. Substantially all refers to at least about 70%, 75%, 80%, 85%, 90% or 95% of the nucleosides are modified. In a further embodiment, all of tlie nucleotides of the sense strand are modified nucleotides; all of the nucleotides of the antisense strand are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0126] In certain embodiments, the dsRNA compound strands comprises an OH or phosphate (P) at the 5’ end of the strand. In a preferred embodiment, the dsRNA compound comprises a sense strand having an OH at the 5’ end of the strand.

[0127] In certain embodiments, the dsRNA compound comprises a strand comprising at least one phosphorothioate intemucleotide (PS) linkage. In certain embodiments, the dsRNA compound comprises a strand comprising a phosphorothioate intemucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). In certain embodiments, the dsRNA compound comprises a phosphorothioate intemucleotide (PS) linkage adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5’ side, the 3’ side or both sides. In certain embodiments, the dsRNA compound comprises a strand comprising a phosphorothioate intemucleotide (PS) linkage adjacent to 2 nucleosides at the 5’ end of the strand and / or 2 nucleosides at the 3’ end of the strand.

[0128] In certain embodiments, the dsRNA compounds or compositions comprise a dsRNA compound listed in any one of Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the dsRNA compound comprises or is any of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1O37 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). In certain embodiments, the dsRNA compound comprises an siRNA compound. In certain embodiments, the siRNA compound is listed in any one of Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA compound comprises or is any of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5). ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsil 226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77).

[0129] Certain embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand (ATsl226 as shown in Table 14, SEQ ID NO: 75) comprises the formula:

[0130] Certain embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises an antisense strand and an antisense strand forming a duplex, wherein the antisense strand (ATal223 as shown in Table 14, SEQ ID NO: 74) comprises the formula:

[0131] Certain embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises an antisense strand and an antisense strand forming a duplex, wherein the antisense strand (ATal253 as shown in Table 14, SEQ ID NO: 80) comprises the formula:

[0132]

[0133] Certain preferred embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand (ATsl226 as shown in Table 14, SEQ ID NO: 75) comprises the formula:

[0134] , and antisense strand (ATal223 as shown in Table 14, SEQ ID NO: 74) comprises the formula:

[0135]

[0136] Certain preferred embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand (ATsl226 as shown in Table 14, SEQ ID NO: 75) comprises the formula:

[0137] , and antisense strand (ATal253 as shown in Table 14, SEQ ID NO: 80) comprises the formula:

[0138] Certain embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand (ATsl227 as shown in Table 14, SEQ ID NO: 77) comprises the formula:

[0139]

[0140] 35 Certain embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises an antisense strand and an antisense strand forming a duplex, wherein the antisense strand (ATal224 as shown in Table 14, SEQ ID NO: 76) comprises the formula:

[0141] Certain embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises an antisense strand and an antisense strand forming a duplex, wherein the antisense strand (ATal254 as shown in Table 14, SEQ ID NO: 81) comprises the formula:

[0142] Certain preferred embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of INHBE in a cell, wherein the siRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand (ATsl227 as shown in Table 14, SEQ ID NO: 77) comprises the formula:

[0143] , and antisense strand (ATal224 as shown in Table 14, SEQ ID NO: 76) comprises the formula:

[0144] Certain preferred embodiments disclosed herein provide a compound comprising an siRNA compound for inhibiting expression of 1NHBE in a cell, wherein the siRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand (ATsl227 as shown in Table 14, SEQ ID NO: 77) comprises the formula:

[0145] , and antisense strand (ATal254 as shown in Table 14, SEQ ID NO: 81) comprises the formula:

[0146] In certain embodiments, the sense and / or antisense strand of ATsi965, ATsi967, ATsi978, ATsi980, ATsi963, ATsi979, ATsi1O31, ATsi1032, ATsi1O37, ATsi1038, ATsi1 l69, ATsi1170,7 ATsi1 l71, ATsi1172, ATsi1173, ATsi1 l74, ATsi1226, ATsi1227, ATsi1255, or ATsi1256 is a salt.

[0147] In particular embodiments, the sense and / or antisense strand consist of the nucleotide sequences as described above. dsRNA can be trafficked into target cells by a variety of modalities. In certain embodiments, dsRNA compounds enter cells via viral delivery vectors, lipid-based delivery, polymer-based delivery, and / or conjugate-based delivery.

[0148] In certain embodiments, the dsRNA compound described herein further comprises a conjugate. In certain embodiments, the siRNA compound listed in any one of Tables 2, 6, 8, 10, 12 or 14 comprises a conjugate, The conjugate can be selected from cholesterols, lipids, carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In a preferred embodiment, the conjugate is an N- Acetylgalactosamine-comprising moiety (GalNAc). In an embodiment, the conjugate can be attached to the 3’ end of a sense strand. In a preferred embodiment, the conjugated dsRNA compound is an siRNA compound further conjugated to a GalNAc. In certain embodiments, the GalNac is attached or conjugated to a dsRNA sense strand al its 3’ end via a phosphate (P).

[0149] In certain embodiments, the dsRNA compounds or compositions disclosed herein comprise a salt of the dsRNA compounds. In certain embodiments, the dsRNA compounds or compositions disclosed herein comprise a salt of the siRNA compounds disclosed in the tables hereinbelow. In certain embodiments, the compounds or compositions disclosed herein comprise a salt of the oligonucleotide strands disclosed in the tables hereinbelow.

[0150] In certain embodiments, the dsRNA compounds or compositions comprise a dsRNA compound that inhibits expression of an INHBE target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In certain embodiments, the dsRNA compound or composition that inhibits expression of an INHBE is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the dsRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ IDNOs: 4 and 5), ATsi979 (SEQ IDNOs: 30 and 31), ATsi1O31 (SEQ IDNOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1O37 (SEQ ID NOs: 52 and 53), ATsi1O38 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1l72 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). In certain embodiments, the dsRNA compound is a siRNA compound that inhibits expression of an INHBE target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In certain embodiments, the siRNA compound or composition that inhibits expression of an INHBE is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi 1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77).

[0151] In certain embodiments, a pharmaceutical composition is provided. The composition which is intended for inhibiting expression of a gene encoding INHBE comprises a dsRNA compound, or salt thereof, alone or in combination with a pharmaceutically acceptable carrier, diluent and / or excipient. In certain embodiments, the dsRNA compound is in a buffer solution. The buffer solution can comprise acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). In certain embodiments, the dsRNA compound in the pharmaceutical composition is an siRNA compound, or salt thereof. In certain embodiments, the siRNA compound is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 3.3), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi 1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1O38 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsil 172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77).

[0152] Certain embodiments disclosed herein provide a method of inhibiting INHBE expression in a cell, the method comprising contacting the cell with a dsRNA compound, a salt thereof, or composition disclosed herein in an amount sufficient to inhibit expression of INHBE, thereby inhibiting expression of INHBE in the cell. In certain embodiments, the method further comprises assessing the expression level of INHBE protein and / or mRNA in the cell. In certain embodiments, the dsRNA compound or composition that inhibits expression of INHBE in a cell is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the dsRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31 ), ATsi 1031 (SEQ ID NOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi 1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi 1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi 1256 (SEQ ID NOs: 81 and 77). In certain embodiments, the dsRNA compound or composition that inhibits expression of INHBE in a cell is an siRNA compound. In certain embodiments, the siRNA is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi 1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi! 173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound.

[0153] Certain embodiments of the invention provide a method for treating and / or preventing an INHBE associated disease, disorder and / or condition in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a dsRNA compound or composition disclosed herein in an amount sufficient to inhibit expression of INHBE, whereby inhibiting expression of INHBE in the subject treats and / or prevents the INHBE associated disease, disorder and / or condition in the subject. In certain embodiments, the dsRNA compound or composition that treats and / or prevents the INHBE associated disease, disorder and / or condition in a subject is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the dsRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi 1037 (SEQ ID NOs: 52 and 53), ATsi1O38 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). In certain embodiments, the dsRNA compound or composition that treats and / or prevents the INHBE associated disease, disorder and / or condition in a subject is an siRNA compound. In certain embodiments, the siRNA compound is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA compound comprises any of the nucleotide sequence and chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi 1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi 1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi 1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is obesity. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is Diabetes Type 11. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is fatty liver disease. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is a cardiovascular disease.

[0154] Certain embodiments of the invention provide an assay to determine the level of INHBE inhibition in a sample from a subject. In certain embodiments, the INHBE assay comprises: a) administering a compound or composition disclosed herein to a subject in an amount sufficient to inhibit expression of INHBE; b) removing a sample from a subject; c) determining the amount of INHBE protein present in the sample; thereby determining the amount of INHBE inhibition by the compound or composition. In certain embodiments, the sample is from blood, serum, urine and / or liver. In certain embodiments, the amount of INHBE protein present in the sample is determined by isolating the INHBE protein from the sample, Western Blotting the protein and probing with an INHBE specific monoclonal antibody to assess the amount of INHBE protein present. In certain embodiments, the amount of INHBE protein present in the sample is determined using ELISA.

[0155] Certain embodiments of the invention provide a method for treating and / or preventing an INHBE associated disease, disorder and / or condition in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any of the dsRNA compounds listed in Tables 2, 6, 8, 10, 12 or 14, thereby treating and / or preventing the INHBE associated disease, disorder and / or condition in the subject. In a preferred embodiment, the dsRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ I D NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ IDNOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1O38 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsil 172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In certain embodiments, the pharmaceutical composition that treats the and / or prevents INHBE associated disease, disorder and / or condition in a subject comprises an siRNA compound. In certain embodiments, the siRNA compound is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsil 172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is obesity. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is Diabetes Type II. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is fatty liver disease. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is a cardiovascular disease.

[0156] In certain embodiments, a symptom of an INHBE associated disease, disorder and / or condition is treated, ameliorated and / or prevented in a subject comprising administering to the subject in need thereof a therapeutically effective amount of a compound comprising or being any of the dsRNA compounds listed in Tables 2, 6, 8, 10, 12 or 14, or a pharmaceutical composition comprising any of the dsRNA compounds listed in Tables 2, 6, 8, 10, 12 or 14. Tn a preferred embodiment, the dsRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsil038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsil 171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In certain embodiments, the compound or pharmaceutical composition that treats, ameliorated and / or prevents the symptom of the INHBE associated disease, disorder and / or condition in the subject is an siRNA compound. In certain embodiments, the siRNA compound is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41 ), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77).

[0157] In certain embodiments, reducing INHBE expression in the liver of a subject has the potential to treat INHBE related diseases, disorder and / or conditions including, but not limited to, metabolic, cardiovascular, hypertensive, inflammatory, cancerous, neurodegenerative, reproductive and / or hepatosteatotic diseases, disorders and / or conditions.

[0158] In certain embodiments, the INHBE associated disease, disorder and / or condition is a metabolic disease, disorder and / or condition. In certain embodiments, the metabolic disease, disorder and / or condition includes, but is not limited to, obesity, adiposity (e.g., abdominal adiposity), dyslipidemia, glucose intolerance, elevated glucose levels (e.g., elevated blood glucose), metabolic syndrome, pre-diabetes, diabetes type 2 (T2DM), insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT), polycystic ovarian syndrome (PCOS) related to insulin resistance, abnormal glycogen metabolism, and overfeeding induced metabolic dysregulation. Some examples of dyslipidemia include primary dyslipidemia (e.g., familial hypercholesterolemia (FH), familial combined or hyperlipidemia (FCHL)) and secondary dyslipidemia (e.g., hyperlipidemia, hypercholesterolemia, or hypertriglyceridemia). Many of the metabolic diseases, disorders and / or conditions also have an inflammatory component and can thus be considered an inflammatory' disease, disorder and / or condition. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is obesity. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is diabetes type II (T2DM).

[0159] In certain embodiments, the INHBE associated disease, disorder and / or condition is a cardio vascular disease, disorder and / or condition. In certain embodiments, the cardiovascular disease, disorder and / or condition includes, but is not limited to, atherosclerosis (e.g., aortic atherosclerosis), coronary artery disease (CAD), peripheral artery disease (PAD), carotid artery disease, cerebrovascular disease, embolisms, ischemic events, restenosis, and / or strokes. Many of the cardiovascular diseases, disorders and / or conditions also have an inflammatory component and can thus be considered an inflammatory disease, disorder and / or condition.

[0160] In certain embodiments, the INHBE associated disease, disorder and / or condition is a hypertensive disease, disorder and / or condition. In certain embodiments, the hypertensive disease, disorder and / or condition includes, but is not limited to, primary hypertension, secondary hypertension and resistant hypertension. Inflammation is an important factor in the development of hypertension; thus, hypertension can be considered an inflammatory disease, disorder and / or condition.

[0161] In certain embodiments, the INHBE associated disease, disorder and / or condition is a hepatosteatotic (also known as fatty liver or hepatic steatosis) disease, disorder and / or condition. In certain embodiments, the hepatosteatotic disease, disorder and / or condition includes, but is not limited to, nonalcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Late stage fatty liver disease may lead to fibrosis and then cirrhosis of the liver. Hepatic steatosis can lead to inflammation, thus, a hepatosteatotic disease, disorder and / or condition can be considered an inflammatory disease, disorder and / or condition. In a preferred embodiment, the INHBE associated disease, disorder and / or condition that would benefit from reduction in INHBE expression is fatty liver disease. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is NAFLD. In a preferred embodiment, the INHBE associated disease, disorder and / or condition is NASH.

[0162] In certain embodiments, the INHBE associated disease, disorder and / or condition is a cancer. In certain embodiments, the cancer is liver cancer or gonadal cancer.

[0163] In certain embodiments, the subject in need of therapeutic treatment with a dsRNA compound or composition disclosed herein is a human subject. In certain embodiments, the subject is overweight or obese. In certain embodiments, the subject has a body mass index (BMI) greater than about 25, 26, 27, 28, 29, or 30.

[0164] Certain embodiments of the invention provide a method for treating a subject having an INHBE associated disease, disorder and / or condition that would benefit from reduction in 1NHBE expression, comprising administering to the subject in need thereof a therapeutically effective amount of a compound comprising or being the dsRNA compound listed in Tables 2, 6, 8, 10, 12 or 14, or a pharmaceutical composition comprising any of the dsRNA compounds listed in Tables 2, 6, 8, 10, 12 or 14, thereby treating the subject having the disorder that would benefit from reduction in INHBE expression. In a preferred embodiment, the dsRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1O37 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi 1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi 1255 (SEQ ID NOs: 80 and 75), or ATsi 1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In certain embodiments, the dsRNA compound that treats the subject having the INHBE associated disease, disorder and / or condition is an siRNA compound. In certain embodiments, the siRNA compound is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi 1031 (SEQ ID NOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1O37 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsil 173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In certain embodiments, the INHBE associated disease, disorder and / or condition is a metabolic, cardiovascular, hypertensive, inflammatory, cancerous, neurodegenerative, and / or hepatosteatotic disease, disorder and / or condition. In a preferred embodiment, the INHBE associated disease, disorder and / or condition that would benefit from reduction in INHBE expression is obesity. In a preferred embodiment, the INHBE associated disease, disorder and / or condition that would benefit from reduction in INHBE expression is fatty liver disease. In a preferred embodiment, the INHBE associated disease, disorder and / or condition that would benefit from reduction in INHBE expression is NASH, In a preferred embodiment, the INHBE associated disease, disorder and / or condition that would benefit from reduction in INHBE expression is dyslipidemia (e.g. hyperlipidemia). In a preferred embodiment, the INHBE associated disease, disorder and-'or condition that would benefit from reduction in INHBE expression is Type 2 diabetes.

[0165] Certain embodiments of the invention provide a method of preventing or reducing at least one symptom in a subject having an INHBE associated disease, disorder and / or condition that would benefit from reduction in INHBE expression, comprising administering to the subject in need thereof a prophylactically effective amount of a compound comprising or being the dsRNA compound listed in Tables 2, 6, 8, 10, 12 or 14, or a pharmaceutical composition comprising any of the dsRNA compounds listed in Tables 2, 6, 8, 10, 12 or 14, thereby preventing or reducing at least one symptom in the subject having the disorder that would benefit from reduction in INHBE expression. In a preferred embodiment, the dsRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsiI037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsil 171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsil 255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In certain embodiments, the dsRNA compound is an siRNA compound. In certain embodiments, the siRNA compound is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13). ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5). ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1O32 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1O38 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsil 171 (SEQ ID NOs: 52 and 68), ATsil 172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Also contemplated are salt forms of the strands comprising the dsRNA compound. In certain embodiments, the at least one symptom is a symptom associated with the INHBE associated disease, disorder / condition, such as inflammation, high blood glucose, adiposity, fatty liver and the like.

[0166] Certain embodiments provide a method for inhibiting the expression of INHBE in a subject comprising the step of administering the compound or composition comprising any of the dsRNA compounds described herein to the subject, in an amount sufficient to inhibit INHBE expression. The dsRNA compound is administered subcutaneously or intravenously to the subject. In certain embodiments, the dsRNA compound inhibits expression of an INHBE target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In certain embodiments, the dsRNA compound is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg. In certain embodiments, the dsRNA compound is a siRNA compound that inhibits expression of an INHBE target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%. In certain embodiments, the dsRNA compound is an siRNA compound that is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg. In certain embodiments, a preferred dose is selected from any of 700mg, 800mg and 900mg. In certain embodiments, a therapeutically effective amount of the compound or composition comprising the dsRNA compound described herein is dosed at about 150mg, 300mg or 600mg once every 3 months. In certain embodiments, a therapeutically effective amount of the compound or composition comprising the dsRNA compound described herein is dosed at about 150mg, 300mg or 600mg once every 6 months. Also contemplated are salt forms of the strands comprising the dsRNA compound.

[0167] In certain embodiments, after administration of the dsRNA compound or composition comprising the dsRNA compound to a subject, the level of INHBE in a sample(s) from the subject is determined, In certain embodiments, the level of INHBE in the subject sample(s) is an INHBE nucleic acid level or protein level in a blood, plasma, urine or liver tissue sample(s). In certain embodiments, the dsRNA compound is an siRNA compound.

[0168] In certain embodiments, the dsRNA compound or composition comprising the dsRNA compound is administered alone or in combination with a second, or additional therapeutic, agent(s) to a subject for treatment of an INHBE associated disease, disorder and / or condition, or symptom thereof. In certain embodiments, the dsRNA compound is an siRNA compound listed in Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the second, or additional therapeutic, agenl(s) is selected from the group consisting of metformin, a thiazolidinedione (TZD), a statin, a glucagon- like peptide 1 (GLP-1) agonist, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a thyroid hormone receptor beta (NR1 A2) agonist, insulin, a fibrate, fish oil, a statin, an adrenergic receptor antagonist, a nitrate (a vasodilator), a diuretic, an anticoagulant, an ACE inhibitor, an angiotensin receptor blocker (ARB), renin inhibitor, calcium channel blocker, adrenergic agonist, anti- hypertensive drug, digoxin, ivabradine, a nonsteroidal anti-inflammatory drug (NS AID), a corticosteroid or is a combination of the therapeutic agents.

[0169] In certain embodiments, the GLP-1 agonist is any of dulaglutide, albiglutide, liraglutide (e.g., Saxenda®), semaglutide (e.g., Wegovy®, Ozempic®, Rybelsus®), exenatide, lixisenatide, tirzepatide and the like. In certain embodiments, the thyroid hormone receptor beta (NR!A2) agonist is resmetirom (Rezdiffra ™).

[0170] In certain embodiments, the thiazolidinedione (TZD) is any of sitagliptin (Januvia®), Pioglitazone (Actos®), Rosiglitazone (Avandia®), and the like. In certain embodiments, the SGLT2 inhibitor is empagliflozin (Jardiance®).

[0171] In certain embodiments, the statin is any of atorvastatin, pravastatin, simvastatin, and the like.

[0172] In certain embodiments, the adrenergic receptor antagonist is a beta blocker (e.g., bisoprolol, nebivolol, atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol or timolol), alpha blocker (e.g., doxazosin, phentolamine, indoramin, phenoxybenzamine, prazosin, terazosin or tolazoline) or mixed alpha + beta blocker (e.g., bucindolol, carvedilol or labelalol), or the like.

[0173] In certain embodiments, the diuretic is a loop diuretic (e.g., bumetanide, ethacrynic acid, furosemide, torsemide), thiazide diuretic (e.g.. epitizide, hydrochlorothiazide, chlorothiazide or bendroflumethiazide), thiazide-like diuretic (e.g., indapamide, chlorthalidone or metolazone), potassium-sparing diuretic (also known as aldosterone receptor antagonist (ARA), e.g., eplerenone, amiloride, triamterene or spironolactone) or the like.

[0174] In certain embodiments, the ACE inhibitor is captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, trandolapril, benazepril or the like.

[0175] Tn certain embodiments, the angiotensin receptor blocker (ARB) is an angiotensin II receptor antagonist such as candesartan, eprosartan, irbesartan, losartan, olmesartan, lelmisartan, valsartan, or the like.

[0176] In certain embodiments, the renin inhibitor is aliskiren or the like.

[0177] In certain embodiments, the calcium channel blocker is a dihydropyridine (e.g., amlodipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine or nitrendipine) or a non-dihydropyridine (e.g., diltiazcm or verapamil).

[0178] In certain embodiments, the vasodilator is sodium nitroprusside or hydralazine or its derivatives.

[0179] In certain embodiments, the adrenergic agonist is an alpha-2 agonist (e.g., clonidine, guanabenz, methyldopa or moxonidine).

[0180] In certain embodiments, the anti-hypertensive drug is guanethidine, reserpine or the like.

[0181] In certain embodiments, the NSAID is ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin or the like.

[0182] In certain embodiments, the corticosteroid is cortisone, hydrocortisone, prednisone, methylprednisolone, dexamethasone, budesonide, triamcinolone acetonide, fluticasone propionate, Betamethasone, Beclometasone or the like.

[0183] In certain embodiments, the additional therapeutic agent(s), when used in combination with the dsRNA compounds or compositions comprising the dsRNA compound described herein, may provide a synergistic or additive effect in treating an 1NHBE associated disease, disorder and / or condition.

[0184] In one embodiment, a process for preparing a sense and / or antisense strand of a double- stranded ribonucleic acid (dsRNA) compound is provided, wherein the process comprises the steps of: a) preparing the sense and / or antisense strand by sequential coupling of modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis on a solid support; b) optionally, coupling an N-Acetylgalactosamine-comprising moiety (GalNAc) to the sense and / or antisense strand on the solid support via the phosphoramidite oligonucleotide synthesis; c) detaching the sense and / or antisense strand from the solid support and removing the solid support; and d) optionally, further purifying the sense and / or antisense strand, optionally using chromatography.

[0185] In one embodiment, a process for preparing a sense and / or antisense strand of a double- stranded ribonucleic acid (dsRNA) compound is provided, wherein the process comprises the steps of: a) coupling a moiety to a solid support via the phosphoramidite oligonucleotide synthesis, b) coupling a modified and / or unmodified nucleotide via the phosphoramidite oligonucleotide synthesis to the moiety on the solid support; c) sequentially coupling additional modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis to prepare die sense and / or anisense strand; d) detaching the sense and / or antisense strand from the solid support and removing the solid support; and e) optionally, further purifying the sense and / or antisense strand, optionally using chromatography. In certain embodiments, the moiety coupled to the solid support is selected from GalNAc, cholesterol, lipid, carbohydrate, phospholipid, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, dye, and the like. In a preferred embodiment, the moiety coupled to the solid support is an N -Acetylgalactosamine- comprising moiety (GalNAc).

[0186] In one embodiment, a process for preparing a sense and / or antisense strand of a double- stranded ribonucleic acid (dsRNA) compound is provided, wherein the process comprises the steps of: a) coupling an N-Acetylgalactosamine-comprising moiety (GalNAc) to a solid support via the phosphoramidite oligonucleotide synthesis, b) coupling a modified and / or unmodified nucleotide via the phosphoramidite oligonucleotide synthesis to the GalNAc on the solid support; c) sequentially coupling additional modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis to prepare the sense and / or anisense strand; d) detaching the sense and / or antisense strand from die solid support and removing the solid support; and e) optionally, further purifying the sense and / or antisense strand, optionally using chromatography.

[0187] In one embodiment, a process of preparing a double-stranded ribonucleic acid (dsRNA) compound is provided, comprising: a) contacting die sense strand prepared according any one of the processes described herein with the antisense strand prepared according any one of the processes described herein in equimolar concentrations in a solution; b) optionally heating the solution to a temperature of about 94°C; and c) optionally reducing the temperature of the solution to about 25°C. The present invention also provides kits comprising any of the dsRNA compounds or any of the pharmaceutical compositions disclosed herein, and optionally, instructions for use. The present invention provides a vial comprising any of the compoimds or any of the pharmaceutical compositions disclosed herein. The present invention provides a syringe comprising any of the compounds or any of the pharmaceutical compositions disclosed herein. In one embodiment, the invention provides a kit for performing a method of inhibiting expression of an INHBE gene in a subject by administering to the subject in need thereof an amount effective to inhibit expression of the INHBE in the subject. The kit comprises a dsRNA compound and instructions / label for use and, optionally, means for administering the dsRNA compound to a subject. In certain embodiments, the compound or pharmaceutical composition is a dsRNA compound listed in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the dsRNA compound comprises or is anyone of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs: 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsi1169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsi1173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). In certain embodiments, the dsRNA compound is an siRNA compound. In certain embodiments, the siRNA compound is as provided in Tables 2, 6, 8, 10, 12 or 14. In a preferred embodiment, the siRNA compound comprises or is any one of the nucleotide sequences with chemical modifications of ATsi965 (SEQ ID NOs: 8 and 9), ATsi967 (SEQ ID NOs: 12 and 13), ATsi978 (SEQ ID NOs: 28 and 29), ATsi980 (SEQ ID NOs: 32 and 33), ATsi963 (SEQ ID NOs: 4 and 5), ATsi979 (SEQ ID NOs: 30 and 31), ATsi1031 (SEQ ID NOs: 40 and 41), ATsi1032 (SEQ ID NOs: 42 and 43), ATsi1037 (SEQ ID NOs; 52 and 53), ATsi1038 (SEQ ID NOs: 54 and 55), ATsil 169 (SEQ ID NOs: 42 and 66), ATsi1170 (SEQ ID NOs: 50 and 67), ATsi1171 (SEQ ID NOs: 52 and 68), ATsi1172 (SEQ ID NOs: 54 and 69), ATsil 173 (SEQ ID NOs: 58 and 70), ATsi1174 (SEQ ID NOs: 62 and 71), ATsi1226 (SEQ ID NOs: 74 and 75), ATsi1227 (SEQ ID NOs: 76 and 77), ATsi1255 (SEQ ID NOs: 80 and 75), or ATsi1256 (SEQ ID NOs: 81 and 77). Certain embodiments provide a double-stranded ribonucleic acid (dsRNA) compound as described herein for use in medicine.

[0188] Certain embodiments provide a double-stranded ribonucleic acid (dsRNA) compound described herein for use in treating, ameliorating, and / or preventing a INHBE associated disease, disorder and / or condition in a subject.

[0189] Embodiment 1 provides a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell, wherein the dsRNA compound comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the antisense strand comprises or is any of the antisense sequences in any one of Tables 2, 6, 8, 10, 12 or 14.

[0190] Embodiment 2 provides a double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell, wherein the dsRNA compound comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the sense strand comprises any of the sense sequences in any one of Tables 2, 6, 8, 10, 12 or 14.

[0191] Embodiment 3 provides the double-stranded ribonucleic acid (dsRNA) compound of embodiment 1 or 2, wherein the dsRNA compound comprises the sense strand of embodiment 2 and the antisense strand of embodiment 1.

[0192] Embodiment 4 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-3, wherein the dsRNA compound is an shRNA compound or an siRNA compound.

[0193] Embodiment 5 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-4, wherein the dsRNA comprises at least one modified nucleotide.

[0194] Embodiment 6 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-5, wherein substantially all of the nucleotides of the sense strand are modified nucleotides; substantially all of the nucleotides of the antisense strand are modified nucleotides; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0195] Embodiment 7 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-6, wherein all of the nucleotides of the sense strand arc modified nucleotides; all of the nucleotides of the antisense strand are modified nucleotides; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. Embodiment 8 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-7, wherein the strand comprises at least one phosphorothioate intemucleotide (PS) linkage.

[0196] Embodiment 9 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-8, wherein the strand comprises a phosphorothioate intemucleotide (PS) linkage adjacent to a deoxyribonucleoside (D) or ribonucleoside (R).

[0197] Embodiment 10 provides the double-stranded ribonucleic acid (dsRNA) compound of embodiment 9, wherein the phosphorothioate intemucleotide (PS) linkage is adjacent to the deoxyribonucleoside (D) or ribonucleoside (R) on the 5’ side, the 3’ side or both sides.

[0198] Embodiment 11 provides the double-stranded ribonucleic acid (dsRNA) compound of embodiment 8, wherein the strand comprises a phosphorothioate intemucleotide (PS) linkage adjacent to 2 nucleosides at the 5* end of the strand and / or 2 nucleosides at the 3 ’ end of the strand.

[0199] Embodiment 12 provides the double-stranded ribonucleic acid (dsRNA) compound of any of the preceding embodiments 1-11 which is an siRNA comprising any one of siRNAs in any one of Tables 2, 6, 8, 10, 12 or 14.

[0200] Embodiment 13 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-12, further comprising a conjugate.

[0201] Embodiment 14 provides the double-stranded ribonucleic acid (dsRNA) compound of embodiment 13, wherein the conjugate is selected from cholesterols, lipids, carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, peptides, dyes.

[0202] Embodiment 15 provides the double-stranded ribonucleic acid (dsRNA) compound of embodiment 13, wherein the conjugate is aann N-Acetylgalactosamine-comprising moiety (GalNAc).

[0203] Embodiment 16 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 13-15, wherein the conjugate is attached to the 3’ end of the sense strand.

[0204] Embodiment 17 provides the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1 -16, wherein the dsRNA compound inhibits expression of an INHBE target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.

[0205] Embodiment 18 provides a pharmaceutical composition for inhibiting expression of a gene encoding INHBE comprising the double-stranded ribonucleic acid (dsRNA) compound of any preceding embodiments 1-17, alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0206] Embodiment 19 provides the pharmaceutical composition of embodiment 18, wherein the dsRNA compound is in a buffer solution.

[0207] Embodiment 20 provides the pharmaceutical composition of embodiment 19, wherein the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

[0208] Embodiment 21 provides the pharmaceutical composition of embodiment 20, wherein the buffer solution is phosphate-buffered saline (PBS).

[0209] Embodiment 22 provides a method of treating and / or preventing an INHBE associated disease, disorder and / or condition in a subject, comprising administering to the subject a therapeutically effective amount of the dsRNA compound or pharmaceutical composition comprising the dsRNA compound of any preceding embodiments 1-21, thereby treating and / or preventing the INHBE associated disease, disorder and / or condition in the subject.

[0210] Embodiment 23 provides the method of embodiment 22 wherein the INHBE associated disease, disorder and / or condition is selected from the group consisting of metabolic, cardiovascular, hypertensive, inflammatory, cancerous, neurodegenerative, and / or hepatosteatotic diseases, disorders and / or conditions.

[0211] Embodiment 24 provides the method of embodiment 22, wherein the hepatosteatotic disease is nonalcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0212] Embodiment 25 provides the method of embodiment 22, wherein the subject is overweight or obese.

[0213] Embodiment 26 provides the method of any one of embodiments 22-25, wherein the dsRNA compound inhibits the expression of INHBE RNA by at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.

[0214] Embodiment 27 provides a method of treating a subject having a disease, disorder and / or condition that would benefit from reduction in INHBE expression, comprising administering to the subject a therapeutically effective amount of the dsRNA compound of any one of embodiments 1-17, or the pharmaceutical composition of any one of embodiments 18-21, thereby treating the subject having the disease, disorder and / or condition that would benefit from reduction in INHBE expression. Embodiment 28 provides a method of preventing at least one symptom in a subject having a disease, disorder and / or condition that would benefit from reduction in INHBE expression, comprising administering to the subject a prophylactically effective amount of the dsRNA compound of any one of embodiments 1-17, or the pharmaceutical composition of any one of embodiments 18-21, thereby preventing at least one symptom in the subject having the disease, disorder and / or condition that would benefit from reduction in INHBE expression.

[0215] Embodiment 29 provides the method of embodiment 27 or 28, wherein the disease, disorder and / or condition is an INHBE associated disease, disorder and / or condition.

[0216] Embodiment 30 provides the method of embodiment 29, wherein the INHBE associated disease, disorder and / or condition is selected from the group consisting of metabolic, cardiovascular, hypertensive, inflammatory, cancerous, neurodegenerative, and / or hepatosteatotic diseases, disorders and-'or conditions.

[0217] Embodiment 31 provides the method of embodiment 30, wherein tlie hepatosteatotic disease, disorder and / or condition is nonalcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0218] Embodiment 32 provides the method of embodiment 31, wherein the subject is overweight or obese.

[0219] Embodiment 33 provides the method of any one of embodiments 22-32, wherein the subject is an animal, preferably a human.

[0220] Embodiment 34 provides the method of any one of embodiments 22-33, wherein tlie dsRNA compound is administered to the subject at a dose of about 0.01 nig / kg to about 50 mg / kg.

[0221] Embodiment 35 provides the method of any one of embodiments 22-34, wherein the dsRNA compound is administered to the subject subcutaneously.

[0222] Embodiment 36 provides the method of any one of embodiments 22-35, further comprising determining the level of INHBE in a sample(s) from the subject.

[0223] Embodiment 37 provides the method of embodiment 36, wherein the level of INHBE in the subject sample(s) is an INHBE nucleic acid level or protein level in a blood, plasma, urine or liver tissue sample(s).

[0224] Embodiment 38 provides the method of any one of embodiments 22-37, further comprising administering to the subject an additional therapeutic agent for treatment of an INHBE associated disease, disorder and / or condition. Embodiment 39 provides the method of embodiment 38, wherein the additional therapeutic agent is selected from the group consisting of metformin, a thiazolidinedione (TZD), a statin, a glucagon-like peptide 1 (GLP-1) agonist, a sodium-glucose co-transporter 2 (SGLT2) inhibitor, a thyroid hormone receptor beta (NR1 A2) agonist, insulin, a fibrate, fish oil, a statin, an adrenergic receptor antagonist, a nitrate (a vasodilator), a diuretic, an anticoagulant, an ACE inhibitor, an angiotensin receptor blocker (ARB), renin inhibitor, calcium channel blocker, adrenergic agonist, anti-hypertensive drug, digoxin, ivabradine, a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid or a combination of the therapeutic agents.

[0225] Embodiment 40 provides a kit comprising the dsRNA compound of any one of embodiments 1-17, or the pharmaceutical composition of any one of embodiments 18-21, and optionally, a label.

[0226] Embodiment 41 provides a process for preparing the sense and / or antisense strand of the double-stranded ribonucleic acid (dsRNA) compound of any one of embodiments 1-17, wherein the process comprises the steps of: a) preparing the sense and / or antisense strand by sequential coupling of modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis on a solid support; b) optionally, coupling an N-Acetylgalactosamine-comprising moiety (GalNAc) to the sense and / or antisense strand on the solid support via the phosphoramidite oligonucleotide synthesis; c) detaching the sense and / or antisense strand from the solid support and removing the solid support; and, d) optionally, further purifying the sense and / or antisense strand, optionally using chromatography.

[0227] Embodiment 42 provides a process for preparing the sense and / or antisense strand of the double-stranded ribonucleic acid (dsRNA) compound of any one of embodiments 1-17, wherein the process comprises the steps of: a) coupling an N-Acetylgalactosamine-comprising moiety (GalNAc) to a solid support via the phosphoramidite oligonucleotide synthesis; b) coupling a modified and / or unmodified nucleotide via the phosphoramidite oligonucleotide synthesis to the GalNAc on the solid support; c) sequentially coupling additional modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis to prepare the sense and / or antisense strand; d) detaching the sense and / or antisense strand from the solid support and removing the solid support; and, e) optionally, further purifying the sense and / or antisense strand, optionally using chromatography. Embodiment 43 provides a process of preparing the double-stranded ribonucleic acid (dsRNA) compound of any one of embodiments 1-17, comprising: a) contacting the sense strand prepared according to claim 41 or 42 with the antisense strand prepared according to embodiments 41 or 42 in equimolar concentrations in a solution; b) optionally heating the solution to a temperature of about 94°C; and, c) optionally reducing the temperature of the solution to about 25°C.

[0228] Embodiment 44 provides a double-stranded ribonucleic acid (dsRNA) compound as defined in any of embodiments 1-17 for use in medicine.

[0229] Embodiment 45 provides a double-stranded ribonucleic acid (dsRNA) compound as defined in any of embodiments 1-17 for use in treating and-'or preventing an INHBE associated disease, disorder and / or condition in a subject.

[0230] The following description applies to all of the above embodiments.

[0231] Oligomeric Compounds line double-stranded RNA (dsRNA) compounds comprise or are oligomeric compounds such as short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs). Presently, they target the INHBE gene by targeting the INHBE mRNA. Oliogmeric compounds may be single- or double-stranded. A dsRNA oligomeric compound of the invention comprises an “antisense strand” to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

[0232] In certain embodiments, an oligomeric compound has a nucleobase sequence that, when written in the 5’ to 3’ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. For example, in certain such embodiments, an siRNA compound comprises an antisense strand which has a nucleobase sequence that, when written in the 5’ to 3’ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

[0233] In certain embodiments, a dsRNA oligomeric compound is 12-30 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 18 to 30 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 12 to 22 subunits in length, In certain embodiments, a dsRNA oligomeric compound is 14 to 30 subunits in length, In certain embodiments, a dsRNA oligomeric compound is 14 to 21 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 15 to 30 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 15 to 21 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 16 to 30 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 16 to 21 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 17 to 30 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 17 to 21 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 18 to 30 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 18 to 21 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 20 to 30 subunits in length, In certain embodiments, a dsRNA oligomeric compound is 15 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 16 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 17 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 18 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 20 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 21 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 22 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 23 subunits in length. Tn certain embodiments, a dsRNA oligomeric compound is 25 subunits in length. In certain embodiments, a dsRNA oligomeric compound is 25 subunits in length. In other embodiments, a dsRNA oligomeric compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the dsRNA oligomeric compounds are 8, 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, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments, the dsRNA oligomeric compound is an siRNA compound. The above description applies to one strand of the the dsRNA oligomeric compound.

[0234] It is possible to increase or decrease the length of a dsRNA oligomeric compound, such as an siRNA compound, and / or introduce base mismatch(s) without eliminating activity (US Patent 7,772,203, incorporated-by-reference herein). For example, it is possible to introduce non- canonical base pairings (e.g., A:G, A:C, G:U, 1:U, I: A, I:C) into an oligomeric compound without eliminating activity. In certain embodiments, designing oligomeric compounds with one or more non-canonical base pairings, i.e., mismatch(s), enhances the activity of the oligomeric compound.

[0235] The dsRNA oligomeric compound can comprise a mismatch(es) with the target, between the oligomeric strands within the duplex, or combinations thereof. The mismatch may occur throughout the siRNA such as in the overhang (a portion of the sense or antisense strand at the 5’ and-'or 3’ end of a duplexed siRNA that has no complementary strand) or the duplex portion.

[0236] Oligomeric Compound Motifs

[0237] A motif refers to a pattern of modification of a dsRNA oligomeric compound. Various motifs have been described in the art and are incorporated-by-reference herein (e.g., US Patent 11,203,755; US Patent 10,870,849; EP Patent 1,532,248; US Patent 11,406,716; US Patent 10,668,170; US Patent 9,796,974; US Patent 8,754,201; US Patent 10,837,013; US Patent 7,732,593; US Patent 7,015,315; US Patent 7,750,144; US Patent 8,420,799; US Patent 8,809,516; US Patent 8,796,436; US Patent 8,859,749; US Patent 9,708,615; US Patent 10,233,448; US Patent 10,273,477; US Patent 10,612,024; US Patent 10,612,027; US Patent 10,669,544; US Patent 11,401,517; USSN 2020 / 0031862; or USSN 2016 / 0272970).

[0238] In certain embodiments, dsRNA oligomeric compounds disclosed herein, such as siRNAs, have chemically modified subunits arranged into motifs to confer on to the dsRNA oligomeric compounds beneficial properties including, but not limited to: enhanced inhibitory activity to increase potency; increased binding affinity to increase specificity for a target nucleic acid, thereby limiting off-target effects and-'or increasing safety; or enhanced resistance to degradation by in vivo nucleases thereby increasing stability and / or durability. In certain embodiments, the dsRNA oligomeric compounds are chimeras where the peripheral nucleobases of the dsRNA oligomeric compounds comprise motifs with various modified or unmodified nucleobases so as to confer increased stability, specificity, safety and / or potency, while the central portion of the compound comprises various modified or unmodified nucleobases to serve as substrates for RISC mediated degradation. Each distinct portion can comprise uniform sugar moieties, modified, or alternating sugar moieties. Each portion can comprise a varied pattern of phosphate and-'or phosphorothioate linkages.

[0239] In certain embodiments, the dsRNA oligomeric compounds targeted to an INHBE nucleic acid comprise a sense strand with sequence and chemical modification motif as shown in Tables 2, 6, 8, 10, 12 or 14. In certain embodiments, the dsRNA oligomeric compounds targeted to an INHBE nucleic acid comprise an antisense strand with sequence and chemical modification motif as shown in Tables 2, 6, 8, 10, 12 or 14.

[0240] Target Nucleic Acids, Target Regions and Nucleotide Sequences

[0241] Several embodiments are directed to methods of modulating gene expression by dsRNA inhibition.

[0242] In certain embodiments, a method of inhibiting Inhibin βE (INHBE) gene expression in a cell comprises administering to the cell a dsRNA compound targeted to an mRNA (or its corresponding cDNA) transcript of INHBE (GenBank NM 031479.5, incorporated herein as SEQ ID NO:1).

[0243] Nucleotide sequences and chemical modification motifs of dsRNA compounds targeting the INHBE transcript are shown in e.g., Tables 2, 6, 8, 10, 12 or 14. It is understood that the nucleotide sequence set forth in each SEQ ID NO in the examples contained herein can be independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. Also, in accordance with the practice of the invention, each SEQ ID NO refers to a nucleotide sequence, with or without chemical modifications, independent of a conjugate moiety. As such, siRNA compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an intemucleoside linkage, or a nucleobase or may further comprise a conjugate moiety. siRNA compounds denoted by ARNATAR designations indicate a combination of sequence and motif.

[0244] Hybridization

[0245] In some embodiments, hybridization occurs between a strand of a dsRNA oligomeric compound disclosed herein and an mRNA. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

[0246] In Watson-Crick canonical base pairings, adenine (A) is complementary to thymine (T) in DNA, adenine (A) is complementary to uracil (U) in RNA, and guanine (G) is complementary' to cytosine (C) in both DNA and RNA. Base pairs, or complementary nucleobases, are usually Watson-Crick base pairs (C:G, A:U, A:T), but, non-canonical base pairs such as Hoogsteen base pairs (e.g., A:G, A:U), Wobble base pairs (e.g., G:U, 1:U, 1: A, 1:C, wherein I is hypoxanthine) and the like are also permitted during hybridization of the oligomeric compound to a target nucleic acid or target region. Wobble base pairs in RNAi agents have previously been described (see e.g., US Patent 7,732,593; US Patent 7,750,144).

[0247] Nucleobase complementarity facilitates hybridization of the dsRNA oligomeric compounds described herein to their target nucleic acids with the stronger the pairing (e.g., the more base pairs and / or the stronger the hydrogen bond), the stronger the hybridization of the oligomeric compound to the target. Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by composition of the oligomeric compound to be hybridized.

[0248] Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the oligomeric compounds provided herein are specifically hybridizable with a target mRNA with little to no off-target binding.

[0249] Complementarity

[0250] An oligomeric compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the oligomeric compound can hybridize with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., inhibition of a target nucleic acid, such as an mRNA nucleic acid).

[0251] Non-complementary nucleobases between an oligomeric compound and an mRNA nucleic acid may be tolerated provided that the oligomeric compound remains able to specifically hybridize to a target nucleic acid. Moreover, an oligomeric compound may hybridize over one or more segments of an mRNA nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch, or hairpin structure).

[0252] In certain embodiments, the oligomeric compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an mRNA nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an oligomeric compound with a target nucleic acid can be determined using routine methods. For example, a dsRNA oligomeric compound in which 18 of 20 nucleobases of the antisense strand of the oligomeric compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. Percent complementarity of a dsRNA oligomeric compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerB L AST programs known in the art (Altschul el al., 1990, J. Mol. Biol., 215:403-410; Zhang and Madden, 1997, Genome Res., 7:649-656) and available through the website for the National Center for Biotechnology Information (NCBI, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Percent homology, sequence identity, or complementarity, can be determined by, for example, NCBI Blast (Johnson et al., Nucleic Acids Res. 2008, 36 (Web Server issue): W5-W9).

[0253] In certain embodiments, the dsRNA oligomeric compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, a strand of the dsRNA oligomeric compound may be fully complementary to an mRNA nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of a dsRNA oligomeric compound strand is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20-nucleobase dsRNA oligomeric compound strand is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the dsRNA oligomeric compound strand.

[0254] Fully complementary can also be used in reference to a specified portion of the dsRNA oligomeric compound strand or the nucleic acid target. For example, a 20-nucleobase portion of a 30-nucleobase dsRNA oligomeric compound strand can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20-nucleobase portion of the 30-nucleobase dsRNA oligomeric compound strand is fully complementary to the target sequence, if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20- nucleobase portion of the dsRNA oligomeric compound strand. At the same time, the entire 30 nucleobase dsRNA oligomeric compound strand may or may not be folly complementary to the target sequence, depending on whether the remaining 10 nucleobases of the dsRNA oligomeric compound strand are also complementary to the target sequence.

[0255] The location of a non-complementary nucleobase may be at the 5’ end or 3’ end of the dsRNA oligomeric compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the dsRNA oligomeric compound. When two or more non- complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous.

[0256] In certain embodiments, dsRNA oligomeric compound strands that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than I non-complementary nucleobase(s) relative io a target nucleic acid, such as an mRNA nucleic acid, or specified portion thereof.

[0257] In certain embodiments, dsRNA oligomeric compound strands that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as an mRNA nucleic acid, or specified portion thereof.

[0258] The dsRNA oligomeric compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of a dsRNA oligomeric compound. In certain embodiments, the dsRNA oligomeric compounds, are complementary to at least an 8-nucleobase portion of a target segment. In certain embodiments, the dsRNA oligomeric compounds are complementary to at least a 9-nucleobase portion of a target segment. In certain embodiments, the dsRNA oligomeric compounds are complementary to at least a 10-nucleobase portion of a target segment. In certain embodiments, the dsRNA oligomeric compounds are complementary' to at least an 11 -nucleobase portion of a target segment. In certain embodiments, the dsRNA oligomeric compounds are complementary to at least a 12-nucleobase portion of a target segment. In certain embodiments, the dsRNA oligomeric compounds are complementary to at least a 13-nucleobase portion of a target segment. In certain embodiments, the dsRNA oligomeric compounds are complementary to at least a 14-nucleobase portion of a target segment. In certain embodiments, the dsRNA oligomeric compounds are complementary to at least a 15- nucleobase portion of a target segment. Also contemplated are dsRNA oligomeric compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

[0259] Chemical Modifications

[0260] A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a covalent linkage (e.g., phosphate group or a chemically modified linkage as described intra) to the sugar portion of the nucleoside. Oligonucleotides are formed through the covalent linkage of adjacent nucleotides to one another, to form a linear sequence of linked nucleotides. Within the oligonucleotide structure, the linkage groups are commonly referred to as forming the intemucleoside linkages of the oligonucleotide. Oligomeric compounds are made up of one (e.g., ssRNAs, antisense oligonucleotides or miRNAs) or more oligonucleotides (e.g., dsRNAs such as siRNAs or shRNAs).

[0261] Modifications to oligomeric compounds encompass substitutions or changes to nucleobases, intemucleoside linkages or sugar moieties. Modified oligomeric compounds as disclosed herein are often preferred over native or unmodified forms because of desirable properties such as, for example, enhanced delivery (e.g., increased cellular uptake), enhanced specificity or affinity for a nucleic acid target, increased stability in the presence of nucleases, enhanced safety (e.g., fewer side effects after administration of the compound to a subject) or increased potency (e.g., inhibitory activity).

[0262] Nucleobase Modifications

[0263] A nucleobase is a heterocyclic moiety capable of base pairing with a nucleobase of another nucleic acid. Modifications to nucleobases can be advantageous to an oligomeric compound for various reasons including, but not limited to, increase stability of the oligomeric compound, increase specificity, decreased immunogenicity of the oligomeric compound, increase affinity of the oligomeric compound, increase potency of the oligomeric compound and / or other desirable features.

[0264] Examples of nucleobase modifications and their advantages are well known in the art (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571 ; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). Nucleobase modifications can comprise substituting nucleobases with nucleobase analogs or modification of a part of the nucleobase. Examples of nucleobase modifications include, but are not limited to, pseudouridine, 2 ’-thiouridine, N6’-methyladenosine, and 5’ -methylcytidine, 5’-fluoro-2’-deoxyuridine, N-ethylpiperidine 5’ triazole-modified adenosine, 5 ’-nitroindole, 2 ’,4 ’-difluorotolylribonucleoside, N-ethylpiperidine 7’-EAA triazole- modified adenosine, 6’-phenylpyrrolocytosine and the like.

[0265] In certain embodiments, dsRNA oligomeric compounds targeted to an mRNA nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobases are, for example, deoxyribonucleosides (D) substituted for ribonucleosides (R). In certain embodiments, a modified nucleobase can be a thymine substitution for an uracil. In certain embodiments, multiple nucleobases of a dsRNA oligomeric compound are modified. In certain embodiments, each nucleobase of a dsRNA oligomeric compound is modified.

[0266] Internucleoside Linkage Modifications

[0267] I’he naturally occuring intemucleoside linkage of RNA and DNA is a 3* to 5' phosphodiester linkage. For nucleosides that include a furanose sugar, the phosphate group can be linked to the 2', 3' or 5* hydroxyl moiety of the sugar. Oligomeric compounds having one or more modified, i.e., non-naturally occurring, intemucleoside linkages are often selected over oligomeric compounds having naturally occurring intemucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, decrease toxicity, increased stability and / or durability, decreased degradation and / or other desirable features for an oligomeric compound. Some modified intemucleoside linkages and their advantages are well known in the art (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571 ; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101).

[0268] Oligomeric compounds having modified intemucleoside linkages include intemucleoside linkages that retain a phosphorus atom as well as intemucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates (e.g., 5’- methylphosphonate (5’-MP)), phosphoramidate, and phosphorothioates (e.g., phosphorodithioate Rp isomer (PS,Rp), phosphorodithioate Rp isomer (PS,Sp), 5’ -phosphorothioate (5’-PS))» methoxypropylphosphonate, (S)-5’-C-methyl with Phosphate, peptide nucleic acid (PNA), 5 ’-(E)- vinylphosphonate.

[0269] In certain embodiments, dsRNA oligomeric compounds targeted to an mRNA nucleic acid comprise one or more modified intemucleoside linkages. In certain embodiments, the modified intemucleoside linkages are phosphorothioate (PS) linkages. In certain embodiments, one or more internucleoside linkages of an oligomeric compound are phosphorothioate intemucleoside linkages. In certain embodiments, the PS linkage is adjacent to a deoxyribonucleoside (sometimes referred to as DNA or “D” herein) or a ribonucleoside (sometimes referred to as RNA, “R” or ”r” herein). In certain embodiments, each intemucleoside linkage of an oligomeric compound is a phosphorothioate intemucleoside linkage.

[0270] Sugar Modifications

[0271] Natural sugars are sugar moieties found in DNA (2’-H) or RNA (2’-OH), i.e., 2-deoxy- beta-D-ribofuranose or beta-D-ribofuranose, respectively. Oligomeric compounds provided herein can contain one or more nucleosides wherein the natural sugar moiety has been modified. Such sugar modified nucleosides may impart desirable features such as increased stability, increased durability (e.g., increased half-life), increased binding affinity, decreased off-target effects, decreased immunogenicity, decreased toxicity, increased potency, or some other beneficial biological property to the oligomeric compounds. Sugar modifications and their advantages are known in the art (Friedrich and Aigner, 2022, BioDrugs, 36(5):549-571 ; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101; Chiu and Rana, 2003, RNA, 9: 1034-1048; Choung et al., Biochem Biophys Res Commun, 2006, 342:919-927; Amarzguioui et al., 2003, Nucleic Acids Res, 31 (2):589-595; Braasch et al., 2003, Biochemistry, 42(26):7967-7975; Czaudema et al., 2003, Nucleic Acids Res, 31(11 ):2705-2716; Allerson et al., 2005, J Med Chem, 48:901-904; Layzer et al., 2004, RNA, 10:766-771; Ui-Tei, et al., 2008, Nucleic Acids Res, 36(7):2136-51; Bramsen and Kjems, 2012, Frontiers in Genetics, 3(154): 1 -22; Bramsen et al., 2010, Nucleic Acids Res, 38(17):5761-5773; Muhonen et al., 2007, Chem & Biodiversity, 4:858-873; which are incorporated-by-reference herein). In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings can include, without limitation, addition of substituted groups (e.g., 5’ sugar modifications, 2’ sugar modifications); bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R) (R = H, C1-C12 alkyl or a protecting group); nucleoside mimetic; and / or combinations thereof.

[0272] A 2’-modified sugar refers to a furanosyl sugar modified at the 2' position. A 2’-modified nucleoside refers to a nucleoside comprising a sugar modified at the 2’ position of a furanose ring. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to any of substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2’ modifications are selected from substituents including, but not limited to: O[(CH2)nO]mCH3, O(CH2)nNH2, ()(CH2)nCH3, O(CH2)nONH2, OCH?.C(-O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2’- substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCIb, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, and / or a group for improving the pharmacodynamic properties of an oligomeric compound, and / or other substituents having similar properties.

[0273] Further examples of nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5’-vinyl, 5*-methyl (R or S), 2’-F-5’-methyl, 4’-S, 2’-deoxy- 2’-fluoro (2’-F), 2’-OCH3(2’-O-methyl, 2’-OMe), 2’-O(CH2)2OCH3(2’-O-methoxyethyl, 2’-O- MOE, 2’-MOE), 2'-O-methyl-4-pyridine, phosphorodiamidate morpholino (PMO), tricyclo-DNA (tcDNA), 2’-arabino-fluoro, 2’-O-benzyl, glycol nucleic acid (GNA), and unlocked nucleic acid (UNA) substituent groups, The substituent at the 2’ position can also be selected from any of allyl, amino, azido, thio, O-allyl, O-Cj-Cio alkyl, OCF3, 0(CH2)2SCH3, O(CH2)2-O-N(Rm)(Rn), and O- CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-Cio alkyl. 2’-OMe or 2’-OCH3or 2’-O-methyl each refers to a nucleoside comprising a sugar comprising an -OCH3 group at the T position of the sugar ring. 2’-F refers to a sugar comprising a fluoro group at the 2’ position. 2’-O-methoxyethyl or 2’-0-M0E or 2’-M0E each refers to a nucleoside comprising a sugar comprising an -O(CH2)2O CH3 group at the 2’ position of the sugar ring.

[0274] BNAs refer to modified nucleosides comprising a bicyclic sugar moiety wherein a bridge connecting two carbon atoms of the sugar ring connects the 2’ carbon and another carbon of the sugar ring. Examples of bicyclic nucleosides include, without limitation, nucleosides comprising a bridge between the 4’ and the 2’ ribosyl ring atoms such as in locked nucleic acid (LNA). In certain embodiments, oligomeric compounds provided herein include one or more bicyclic nucleosides wherein the bridge comprises a 4’ to 2’ bicyclic nucleoside. LNAs and UNAs have been described by Campbell and Wengel (Chem Soc Rev, 2011, 40(12):5680-9) and are incorporated-by-reference herein.

[0275] In certain embodiments, dsRNA oligomeric compounds comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety has a 2’-OMe modification. In certain embodiments, the modified sugar moiety has a 2’-F modification. In certain embodiments, the 2’-0Me and / or 2’-F modified nucleotides are arranged in a motif. In a preferred embodiment, the modifications are arranged in ARNATAR motifs as disclosed in WO2024137543, which is incorporated-by-reference herein.

[0276] In certain embodiments, the dsRNA oligomeric compounds targeted to a nucleic acid comprises a sense strand with motif described by the one of the following formulas:

[0277] Formula (I): 5’ M-(Y)n-Z-(Y)r-D-D 3’,

[0278] Formula (II): 5’ Y-Z-(Y)q-FFNM-(Y)q-M-(Y)v-D-D 3’,

[0279] Formula (III): 5’ M*F*MMN*MN*MFFNMN*MN*MMFM*D*D 3’,

[0280] Formula (X): 5’ Ml-MMNMNMFFNMNMNMMNMDD 3’, or

[0281] Formula (XI): 5’ MFMMNMNMFFMMNMNMMFMDD 3’, wherein each D is a deoxyribonucleoside (D is a modification of R), each R is a ribonucleoside, each N is a nucleoside, modified or unmodified (e.g., D, R, M, F, IJNA modified, or LNA modified), each M is a 2’-OMe modified nucleoside, each F is a 2’-F modified nucleoside, each * is a phosphorothioate (PS) linkage, each Y is two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF or FM) or a modified nucleoside adjacent to an unmodified nucleoside (e.g., DR, RD, MR or RM), each Z is two adjacent unmodified nucleosides or two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides (e.g.. MM, DD, RR, or FF), each n is 6-8, each q is 2-3, each r is 1 -2, each v is 0-1, and wherein no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the FFNM is FFRM or FFMM.

[0282] In certain embodiments, the oligomeric compounds targeted to a nucleic acid comprise an antisense strand with motif described by one of the following formulas:

[0283] Formula (IV): 5'-L-M-(D)v-(Y)s-(Z)t-(Y)u-Z-N-(Z)r 3’,

[0284] Formula (V): 5’ L-(Y)p-NM-(FMM)r-(Y)p-(Z)r 3’,

[0285] Formula (VI): 5’ L-M*N*MNMFNMFMMNMFMFMMN*M*M 3’,

[0286] Formula (VII): 5’ L-M*D*MFMFNMFMMFMFMFMMN*M*M 3’,

[0287] Formula (VIII): 5* L-MNMNMFNMFMMNMFMFMMNMM 3’,

[0288] Formula (IX): 5’ L-M-(Y)p-Z-(Y)p-(Z)r 3’, or

[0289] Formula (XII): 5’ L-MDMFMFNMFMMFMFMFMMNMM 3’, wherein each D is a deoxyribonucleoside (D is a modification of R), each R is a ribonucleoside, each N is a nucleoside, modified or unmodified (e.g., D, R, M, F, UNA modified, or LNA modified), each M is a 2’-OMe modified nucleoside, each F is a 2’-F modified nucleoside, each L is a 5’ phosphate, 5’ vinyl phosphonate or 5’ OH each ♦ is a phosphorothioate (PS) linkage, each Y is two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF or FM) or a modified nucleoside adjacent to an unmodified nucleoside (e.g., DR, RD, MR or RM), each Z is two adjacent unmodified nucleosides or two adjacent nucleosides with the same modification or two adjacent unmodified nucleosides (e.g., MM, DD, RR, or FF), each (5p) is 5 ’-phosphate, each n is 6-8, each p is 3-5, each r is 1-2, each v is 0-1, each s is 2-7, each t is 0-2, and wherein no single modification type modifies more than two consecutive nucleotides. In certain embodiments, the FNM is FMM.

[0290] Oligomeric Compound Delivery Systems

[0291] Oligomeric compounds require entry into target cells to become active. A variety of modalities have been used to traffic oligomeric compounds into target cel Is including viral delivery vectors, lipid-based delivery, polymer-based delivery, and conjugate-based delivery (Paunovska et al.. Drug Delivery Systems for RNA Therapeutics, 2022, Nature Reviews Genetics, 23(5):265 - 280; Chen et al., 2022, Molecular Therapy, Nucleic Acids, 29:150-160).

[0292] Lipid-based particles can form specific structures such as micelles, liposomes and lipid nanoparticles (LPNs) to carry oligomeric compounds into cells. To form these particles, LPNs can include one or more of a cationic or ionizable lipid (e.g., DLin-MC3-DMA, SM-102, ALC-0315), cholesterol, a helper lipid, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), polyethylene glycol) (PEG) modified lipid (e.g., PEG-2000-C-DMG, PEG-2000-DMG, ALC-0159), C12-200, cKK-E12 and the like. Different combinations of lipids can be formulated to affect the delivery of the oligomeric compound to different types of cells. In one example, therapeutic siRNA patisiran was formulated in cationic ionizable lipid DLin-MC3-DMA, cholesterol, polar phospholipid DSPC, and PEG-2000-C-DMG for delivery to hepatocytes. Polymer-based particles are also used in oligomeric compound delivery systems. Such polymers include poly(lactic-co-glycolic acid) (PLGA), polyethylenimine (PEI), poly(l-lysine) (PLL), poly(beta-amino ester) (PBAE), dendrimers (e.g., poly(amidoamine) (PAMAM) or PLL), and other polymers or modified polymers thereof. The polymer composition can be varied depending on the trails desired for delivery of the oligomeric compound.

[0293] The dsRNA oligomeric compounds disclosed herein may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting compound. Conjugate groups can include cholesterols, lipids, carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, dyes, etc. Conjugate-based delivery can actively deliver oligomeric compounds to specific cell types.

[0294] In an example, an N-Acetylgalactosamine-comprising moiety (GalNAc) is conjugated to a dsRNA oligomeric compound and delivers it into hepatocytes. Various GalNAc conjugates can be found in several publications including the following, all of which are incorporated-by-reference herein: Sharma et al., 2018, Bioconjugate Chem, 29:2478-2488; Nair et al., J. Am. Chem. Soc. 2014, 136(49): 16958-16961; Ream, 2022, Drugs, 82:1419-1425; US Patent 10,087,208; Prakash et al., 2014, Nucleic Acids Res, 42(13):8796-807; Debacker et al., 2020, Molecular Therapy, 28(8):1759-1771 ; US Patent 11,110,174; US Patent 9,796,756; US Patent 9,181,549; US Patent 10,344,275; US Patent 10,570,169; US Patent 9,506,030; and, US Patent 7,582,744; WO2024137545.

[0295] In certain embodiments, the following GalNAc conjugate precursor with solid support can be used to conjugate an INHBE dsRNA compound at the 3’ end of the sense strand. In a preferred embodiment, the dsRNA compound is an ARNATAR designed siRNA compound selected from Tables 2, 6, 8, 10, 12 or 14.

[0296]

[0297] In a preferred embodiment, the GalNAc conjugated to an oligonucleotide is the GalNAc (also known as AN-GalNAc) shown as follows:

[0298] In one embodiment, AN-GalNAc is conjugated to a sense strand of a dsRNA compound. In a preferred embodiment, AN-GalNAc conjugated to a dsRNA compound selected from any of the compounds in Tables 2, 6, 8, 10, 12 or 14.

[0299] Oligomeric Compound Synthesis siRNAs were designed, synthesized, and prepared using methods known in the art.

[0300] Solid phase syntheses of oligonucleotides were done on a MerMade™ 48x synthesizer (BioAutomation, LGC, Biosearch Technologies, Hoddesdon, UK), which can make up to 48 1 μMole or 5 μMole scale oligonucleotides per run using standard phosphoramidite chemistry. Phosphoramidite synthesis of oligonucleotides on a solid support is well known in the art (e.g., Beaucage and Caruthers, 1981, Tetrahedron Letters, 22(20): 1859-1862; Roy and Caruthers, 2013, Molecules, 18:14268-14284; and Roy and Caruthers et al., 2021, Nature Communications, 12:2760). Solid support is controlled pore glass (500-1400 A) loaded with universal linkers or loaded with 3’-GalNAc conjugates (e.g., AM Chemicals, Vista, CA, USA; Primetech ALC, Minsk, Belarus; Gene Link, Elmsford, NY, USA; or any GalNAc conjugate disclosed herein) or universal solid support (AM Chemicals, Vista, CA, USA). Ancillary synthesis reagents and standard 2’- cyanoethyl phosphoramidite monomers (2’ -fluoro, 2’-O-methyl, RNA, DNA) were obtained from various sources (Hongene Biotech, Shanghai, China; Sigma-Aldrich, St. Louis, MO, USA; Glen Research, Sterling, VA, USA; ThermoFisher Scientific, Waltham, MA, USA; LGC Biosearch Technologies, Hoddesdon, UK). Phosphoramidite mixtures were prepared in anhydrous acetonitrile or 30% DMF:acetonitrile and were coupled using 0.25M 4,5-dicyanoimidazole (DCI) (Sigma- Aldrich, St. Louis, MO, USA) with coupling times ranging from 120-360 seconds. Standard phosphodiester linkages were achieved using 0.02M iodine mixture in Tetrahydrofuran (THF), pyridine and water. Phosphorothiate linkages were generated using 0.05M sulfurizing Reagent II (3-((Dimethylamino-methylidene)amino)-3H-l,2,4-dithiazole-3-thione, DDTT) (40:60, Pyridine / Acetonitrile) (LGC Biosearch Technologies, Hoddesdon, UK) with an oxidation time of 6 minutes. All sequences were synthesized with Dimethoxy Trityl (DMT) protecting group removed.

[0301] Upon completion of solid phase synthesis, the oligonucleotides were cleaved from the solid support and deprotection of base labile groups performed by incubation in ammonium hydroxide at 55'C for 6 hours. Ammonium hydroxide was removed using a centrifugal vacuum concentrator to dryness at room temperature. For sequences containing natural ribonucleotides (2 ’-OH ) protected with tert-butyl dimethyl silyl (TBDMS), a second deprotection was performed using triethylamine; trihydrofluoride (TEA:3HF). To each TBDMS protected oligonucleotide 100 μL DMSO and 125μL TEA:3HF were added and incubated at 65’C for 2.5 hours. After incubation, 25μL of 3M sodium acetate was added to the solution which was subsequently precipitated in butanol at -20'C for 30 minutes. The cloudy solution was centrifuged to a cake at which time the supernatant was carefully decanted with a pipette. The standard precipitation process was then completed with 75% ethanokwater then 100% ethanol as supernatant solutions. The oligonucleotide cake was dried for 30 minutes in a centrifugal vacuum concentrator.

[0302] Desalting without HPLC purification was performed after precipitation with 3M sodium acetate with a follow on G25 Sephadex® column (Sigma-Aldrich, St. Louis, MO, USA) elution. Purification of oligonucleotides was afforded by anion exchange chromatography on a Gilson GX271 prep HPLC system (Middleton, W1, USA) using BioWorks Q40 resin (Uppsala, Sweden). Final desalt was performed by Sephadex® G25 column. All oligonucleotides were analyzed by ion pairing reverse phase HPLC for purity an Agilent 1200 analytical HPLC (Santa Clara, CA, USA), negative ion mass spectrometry for intact mass on an Agilent 6130 single quad mass spectrometer (Santa Clara, CA, USA), and A260 quantification by UV / Vis on a Tecan Infinite® M Plex plate reader (Zurich, Switzerland).

[0303] Double-Stranded Oligomeric Compound Duplex Formation

[0304] In general, for a double-stranded oligomeric compound such as an siRNA compound, a sense and antisense oligonucleotide is annealed together to form a duplex. The duplex is formed by contacting the sense strand prepared according to any one of the processes described herein with the antisense strand prepared according to any one of the processes described herein in equimolar concentrations in a solution. Optionally, the solution is heated to a temperature of about 94°C then the temperature is reduced to about 25°C. In an example, duplex formation of 50- 300mM can be achieved by heating samples at 94°C for 4 mins in lx phosphate-buffered saline in a block heater, followed by removal of the heating block containing the samples from the block heater and allowing it to gradually cool down to room temperature over a time course of Ihr.

[0305] Compositions and Methods for Formulating Pharmaceutical Compositions

[0306] The dsRNA compounds, such as siRNA compounds targeting INHBF. described herein, can be combined with pharmaceutically acceptable active or inert substances, such as a diluent, excipient or carrier, for the preparation of pharmaceutical compositions or formulations.

[0307] Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered. In certain embodiments, the pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acid compounds to an animal. The excipient can be liquid or solid and can be selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0308] Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with nucleic acid compounds, suitable for parenteral or non-parenteral administration can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising a dsRNA compound targeted to an INHBE nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the dsRNA compound is an siRNA compound.

[0309] Pharmaceutical compositions comprising dsRNA compounds such as siRNA compounds can encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other dsRNA compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of dsRNA compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0310] In certain embodiments, a pharmaceutical composition is prepared for administration byinjection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer (e.g., PBS). In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers.

[0311] Dosages

[0312] For purposes of the disclosure, the amount or dose of the active agent (oligomeric compound of the invention) administered should be sufficient to e.g., inhibit the expression of INHBE in an animal, in the animal (e.g., human), dose will be determined by the efficacy of the particular active agent and the condition of the animal, as well as the body weight of the animal to be treated.

[0313] Many assays for determining an administered dose are known in the art.

[0314] The dose of the active agent of the present disclosure will also be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular active agent of the present disclosure. Typically, the attending physician will decide the dosage of the active agent of the present disclosure with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, active agent of the present disclosure to be administered, route of administration, and the severity of the condition being treated.

[0315] Dosing

[0316] In certain embodiments, pharmaceutical compositions are administered according to a dosing regimen (e.g., dose, dose frequency, and duration) wherein the dosing regimen can be selected to achieve a desired effect i.e., is therapeutically effective in a subject. The desired effect can be, for example, reduction of INHBE or the prevention, reduction, amelioration or slowing the progression of a disease, disorder and / or condition, or symptom thereof, associated with INHBE in a subject. In certain embodiments, the variables of the dosing regimen are adjusted to result in a desired concentration of pharmaceutical composition in a subject. "Concentration of pharmaceutical composition" as used with regard to dose regimen can refer to the dsRNA compound or active ingredients) of the pharmaceutical composition. For example, in certain embodiments, dose and dose frequency are adjusted to provide a tissue concentration or plasma concentration of a pharmaceutical composition at an amount sufficient to achieve a desired effect.

[0317] Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Dosing is also dependent on drug potency and metabolism differences between subjects. In certain embodiments, a therapeutically effective dosage in a subject is from about 0.01 μg to 50 mg per kg of body weight, 0.01 μg lolOO mg per kg of body weight, or within a range of about 0.001 mg to 1000 mg dosing, and may be given once or more daily, weekly, monthly, quarterly or yearly, or even once every 2 to 20 years. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the dsRNA compound is administered in maintenance doses, ranging from about 0.01 μg to 100 mg per kg of body weight, once or more daily, once or more weekly, once or more monthly, once or more quarterly, once or more yearly, to once every 20 years or ranging from about 0.001 mg to 1000 mg dose. In certain embodiments, it may be desirable to administer the dsRNA compound from at most once daily, once weekly, once monthly, once quarterly, once yearly, once every two years, once every three years, once every four years, once every five years, once every ten years, to once every 20 years.

[0318] In certain embodiments, the range of therapeutically effective dosing may be between any of about lmg-1500mg, 100mg-1400mg, 100mg-1300mg, 100mg-1200mg, 100mg-l lOOmg, lOOmg-lOOOmg, 100mg-900mg, 200mg-800mg, 300mg-700mg, 400mg-600mg, 100mg-400mg, 200mg-500mg, 300mg-600mg, and 400mg-700mg. In certain embodiments, a therapeutically effective dose is about lOOmg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 800mg, 850mg, 900mg, 950mg, lOOOmg, 1050mg, 1 lOOmg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, or 1500mg. In certain embodiments, a preferred dose is selected from any of about 700mg, 800mg and 900mg. In certain embodiments, a therapeutically effective amount of the dsRNA compound is dosed at any of about 150mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, or 900mg twice a year. In certain embodiments, a therapeutically effective amount of the dsRNA compound is dosed at about 150mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, or 900mg quarterly. In certain embodiments, a therapeutically effective amount of the dsRNA compound is dosed at about 150mg, 300mg or 600mg once every 3 months. In certain embodiments, a therapeutically effective amount of the dsRNA compound is dosed at about 150mg, 300mg or 600mg once every 6 months.

[0319] Administration

[0320] The dsRNA compounds such as siRNA compounds or pharmaceutical compositions of tire present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be oral, inhaled or parenteral.

[0321] In certain embodiments, the dsRNA compounds and compositions as described herein are administered parenterally. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certahi embodiments, infused pharmaceutical agents are delivered with a pump.

[0322] In certain embodiments, parenteral administration is by injection. The injection can be delivered with a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue or organ.

[0323] In certain embodiments, formulations for parenteral, intrathecal or intraventricular administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0324] In certain embodiments, formulations for oral administration of the compounds or compositions can include, but are not limited to, pharmaceutical carriers, excipients, powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In certain embodiments, oral formulations are those in which compounds provided herein are administered in conjunction with one or more penetration enhancers, surfactants and chelators.

[0325] In Vitro Testing of dsRNAs

[0326] Described herein are methods for treatment of cells with dsRNA compounds, for example siRNA compounds, which can be modified appropriately for treatment with other oligomeric compounds.

[0327] Cells may be treated with siRNA compounds when the cells reach approximately 60-80% confluency in culture.

[0328] One reagent commonly used to introduce siRNAs into cultured cells includes the cationic lipid transfection reagent Lipofectamine™ RNAiMAX (Invitrogen, Waltham, MA). siRNAs may be mixed with Lipofectaminei MRNAiMAX in OPTI-MEM 1 (Thermo Fisher Scientific, Waltham, MA) to achieve the desired final concentration of siRNA and a Lipofectamine™ RNAiMAX concentration that may range from about 0.001 to 300 nM siRNAs. Transfection procedures are done according to the manufacturer’s recommended protocols.

[0329] Another technique used to introduce siRNA compounds into cultured cells includes electroporation. siRNA compounds conjugated with an N- Acetylgalactosamine-comprising moiety (GalNAc) can be introduced to cells through incubation of the siRNA compounds with cells without transfection reagents, referenced herein as “free uptake”, The siRNA-GalNAc conjugates are transported into asialoglycoprotein receptor (ASGR) positive cells such as hepatocytes via endocytosis.

[0330] Cells are treated with siRNA compounds by routine methods. Cells may be harvested 4 - 144 hours after siRNA compounds treatment, at which time mRNA (harvested at 4-144 hrs) or protein levels (extracted at 24-96 hrs) of target nucleic acids are measured by methods known in the art and described herein. In general, treatments are performed in multiple replicates, and the data are presented as the average of the replicate treatments plus the standard deviation.

[0331] The concentration of siRNA compounds used varies from cell line to cell line and target to target. Methods to determine the optimal siRNA compounds concentration for a particular target in a particular cell line are well known in the art. In general, cells are treated with siRNA compounds in a dose dependent manner to allow for the calculation of the half-maximal inhibitory concentration value (IC50). siRNA compounds are typically used at concentrations ranging from about 0.001 nM to 300 nM when transfected with Lipofectamine™ RNAiMAX. siRNA compounds are used at higher concentrations ranging from about 625 to 20,000 nM, when transfected using electroporation or free uptake.

[0332] RNA Isolation

[0333] RNA analysis of INHBE mRNA levels can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Thermo Fisher Scientific, Waltham, MA), Qiagen RNeasy kit (Qiagen, Hilden, Germany), or AcroPrep Advance 96-well Filter Plates (Pall Corporation, Port Washington, New York) using Qiagen’s RL'T ", RW1 and RPE buffers (Qiagen, Hilden, Germany). RNA extraction procedures are done according to the manufacturer’s recommended protocols.

[0334] In Vivo Testing of dsRNA Compounds dsRNA compounds, for example, siRNA compounds, are tested in animals to assess their ability to inhibit expression of INHBE and produce phenotypic changes such as a decrease in one or more INHBE related diseases / disorders and / or conditions. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, dsRNAs compounds are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of dsRNA compound dosage and dosing frequency depends upon factors such as route of administration and animal body weight. In one embodiment, following a period of treatment with dsRNA compounds, mRNA encoding INHBE is isolated from liver tissue and changes in INHBE expression are measured. Changes in INHBE protein levels can also be measured. Changes in INHBE expression can also be measured by determining the levels of markers for INHBE, such as b-hydroxybutyrate, Adrb3, Atgl, Cgi58, Hsl and / or Fabp4, in the animal. Certain Indications

[0335] In certain embodiments, the invention provides methods of treating a subject comprising administering one or more compounds and / or pharmaceutical compositions of the present invention to the subject. In certain embodiments, the subject has, or is at risk for, an INHBE related disease, disorder and / or condition, or symptom thereof. In certain embodiments the invention provides methods for prophylactically reducing INHBE expression in a subject. Certain embodiments include treating a subject in need thereof by administering to the subject a therapeutically effective amount of a dsRNA compound such as an siRNA targeted to an INHBE nucleic acid.

[0336] In certain embodiments, administration to a subject of a therapeutically effective amount of a dsRNA compound targeted to an INHBE nucleic acid is accompanied by monitoring of INHBE levels in the blood plasma or tissue of the subject, to determine a subject's response to administration of the dsRNA compound. A subject's response to administration of the dsRNA compound is used by a physician to determine the amount and duration of therapeutic intervention.

[0337] In certain embodiments, administration to a subject of a dsRNA compound targeted to an INHBE nucleic acid results in reduction of INHBE expression by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% or a range defined by any two of these values. In certain embodiments, administration to a subject of a dsRNA compound targeted to an INHBE nucleic acid results in a change in the INHBE related disease, disorder, condition, symptom and / or marker (e.g., hypertension or organ damage) in the subject. In certain embodiments, administration to a subject of an INHBE dsRNA compound increases or decreases the INHBE related disease, disorder, condition, symptom and / or marker by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% or a range defined by any two of these values in the subject.

[0338] In certain embodiments, pharmaceutical compositions comprising a dsRNA compound targeted to INHBE are used for the preparation of a medicament for treating a subject suffering or susceptible to an INHBE related disease, disorder or condition. In certain embodiments, the dsRNA compound is an ARNATAR designed siRNA compound targeting INHBE as listed in Tables 2, 6, 8, 10, 12 or 14. Certain Combination Therapies

[0339] In certain embodiments, a first agent comprising a dsRNA compound provided herein is co-administered with one or more secondary agents to a subject. In certain embodiments, the dsRNA compound is an ARNATAR siRNA compound listed in Tables 2, 6, 8, 10, 12 or 14.

[0340] In certain embodiments, such second agents are designed to treat the same INHBE related disease, disorder and / or condition as the first agent described herein. In certain embodiments, such second agents are designed to treat a different disease, disorder, or condition as the first agent described herein. In certain embodiments, such second agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, such first agents are designed to treat an undesired side effect of a second agent. In certain embodiments, second agents are co-administered with the first agent to treat an undesired effect of the first agent. In certain embodiments, second agents are co-administered with the first agent to produce a combinational or additive effect. In certain embodiments, second agents are co- administered with the first agent to produce a synergistic effect.

[0341] In certain embodiments, the co-administration of the first and second agents permits use of lower dosages than would be required to achieve a therapeutic or prophylactic effect if the agents were administered as independent therapy. In certain embodiments the dose of a co-administered second agent is the same as the dose that would be administered if the second agent was administered alone. In certain embodiments the dose of a co-administered second agent is greater than the dose that would be administered if the second agent was administered alone.

[0342] In certain embodiments, a first agent and one or more second agents are administered at the same time. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical formulation. In certain embodiments, the first agent and one or more second agents are prepared separately.

[0343] In certain embodiments, second agents include, but are not limited to, certain procedures to reduce metabolic, cardiovascular, hypertensive, inflammatory, cancerous, neurodegenerative, and / or hepatosteatotic diseases, disorders and / or conditions. Examples of such procedures include, but are not limited to, diet changes, lifestyle changes (e.g. exercising) and / or surgical procedures (e.g., stent placement, angioplasty, by-pass surgery, bariatric surgery, gastric bypass, intragastric balloon, gastric band, or gastric sleeve). The second agents can be used in combination with the therapeutic compounds described herein to decrease an INHBE related disease, disorder and / or condition in a subject.

[0344] Kits of The Invention

[0345] According to another aspect of tlie invention, kits are provided. Kits according to the invention include package(s) comprising any of the compositions of the invention or oligomeric compound of the invention. In various aspects, the kit comprises any of the compositions of the invention as a unit dose. For purposes herein “unit dose" refers to a discrete amount dispersed in a suitable carrier.

[0346] The phrase "package" means any vessel containing compositions presented herein. In preferred embodiments, the package can be a box or wrapping. Packaging materials for use in packaging pharmaceutical products are well known to those of skill in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including pre-filled syringes), bottles, and any- packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0347] The kit can also contain items that are not contained within the package but are attached to the outside of the package, for example, pipettes.

[0348] Kits may optionally contain instructions for administering compositions of tlie present invention to a subject having a condition in need of treatment. Kits may also comprise instructions for approved uses of components of the composition herein by regulatory agencies, such as the United States Food and Drug Administration. Kits may optionally contain labeling or product inserts for the present compositions. The package(s) and / or any product insert(s) may themselves be approved by regulatory agencies. The kits can include compositions in the solid phase or in a liquid phase (such as buffers provided) in a package. The kits also can include buffers for preparing solutions for conducting the methods, and pipettes for transferring liquids from one container to another.

[0349] The kit may optionally also contain one or more other compositions for use in combination therapies as described herein. In certain embodiments, the package(s) is a container for any of the means for administration such as intravitreal delivery, intraocular delivery, intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, intra-arterial delivery, intraventricular delivery, intrastemal delivery, intracranial delivery, or intradermal injection.

[0350] Methods of Use and Compounds for Use

[0351] The invention provides methods for inhibiting the expression of INHBE in a subject and methods of treating and / or preventing an INHBE associated disease, disorder and / or condition or the symptoms thereof in a subject comprising administering an effective amount of a dsRNA compound of the invention or a pharmaceutical composition of the invention, so as to inhibit the expression of INHBE in the subject. The invention also provides the compounds for use in treating ans / or preventing an INHBE associated disease, disorder and / or condition in a subject.

[0352] In some embodiments of the present disclosure, the subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perissodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order .Anthropoids (humans and apes). In a preferred aspect, the mammal is a human.

[0353] ADVANTAGES OF THE INVENTION

[0354] Disclosed herein are dsRNA compounds (e.g., siRNAs) targeting INHBE improved with Advanced RNA Targeting (ARNATAR) abilities that enhance their gene silencing activity. The dsRNA compounds utilize ARNATAR motifs in conjunction with INHBE targeting sequences to produce stable and / or durable therapeutic compounds allowing longer lasting benefits for acute and / or chronic diseases and / or less frequent dosing of the therapeutic compound. In addition to stability and / or durability, the dsRNA compounds utilizing ARNATAR motifs may have a quicker mode of action (for example, by knocking down INHBE expression at an earlier time than the reference compound), which would be beneficial for acute diseases. In some instances, ARNATAR dsRNA compounds have been found to be more potent than a reference compound.

[0355] Another benefit of ARNATAR designed dsRNA compounds targeting INHBE is a shortness of length. This shortness of length allows a shorter synthesis protocol, shorter synthesis time and / or decreases the cost of manufacturing the compounds.

[0356] Additionally, ARNATAR designed dsRNA compounds are very potent inhibitors of INHBE. The high potency allows INHBE reduction in tissues other than liver.

[0357] Accordingly, there is a need for improved dsRNA compounds to treat diseases. ARNATAR dsRNA compounds targeting INHBE have been designed to improve speed, stability, specificity, safety and / or potency in order to produce an improved therapeutic.

[0358] EXAMPLES

[0359] While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is herein incorporated-by-reference in its entirety.

[0360] EXAMPLE 1: DESIGNING INHBE siRNA WITH VARYING CHEMICAL MOTIFS

[0361] A human Inhibin βE (INHBE) transcript (Table 1 ) was targeted by siRNAs designed with ARNATAR motifs.

[0362] Table 1 : INHBE Target Sequence

[0363] INHBE targeting siRNAs (also known as INHBE siRNAs) were designed as shown in Table 2.

[0364] The following applies to all modified sequences disclosed herein:

[0365] A notation is made before each nucleotide indicating the type of chemical modification, if any, made to the nucleotide. If no modification notation is made before a letter designating a nucleotide, the nucleotide is a deoxyribonucleotide. Notations for the chemical modifications to the strands can be found as follows:

[0366] (5p) = 5* -phosphate r ribonucleotide d (or no notation made before a nucleotide) - deoxyribonucleotide which has been substituted for a ribonucleotide f = 2’-F m - 2’-OMe

[0367] * = phosphorothioate (PS) linkage which has been substituted for a phosphate ( PO) linkage gna = glycol nucleic acid

[0368] If more than one sequence is disclosed in one row of the tables, the SEQ ID NO applies to the modified sequence (“Sequence + Chemistry”).

[0369] In the ARNA'l AR designed siRNA compounds targeting INHBE disclosed herein, a sense strand is conjugated with an N -Acetylgalactosamine-comprising moiety (GalNAc) (AN-GalNAc) as described in WO2024137545 (incorporated-by-reference herein). As a control, a reference siRNA (also known as ATsi972 or benchmark siRNA herein) was synthesized and included in the studies described herein. This reference siRNA ATsi972 (Table 2A) mirrors a compound known as AD- 1708473 in the reference W02023003922 which uses the GalNAc (AL-GalNAc) described therein.

[0370] Table 2. INHBE siRNAs with Modifications to Both Strands

[0371] In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in Hep3B Cells siRNAs described in Table 2 were transfected at 0-10 nM into Hep3B cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 18 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 3. The IC50 of the siRNAs targeting INHBE are shown in Table 4, and the dose responses of siRNAs on INHBE mRNA levels in Hep3B cells are shown in Figure 1. INHBE mRNA levels were determined through qRT-PCR using INHBE specific primer probe sets as listed in Table 3. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The INHBE target RNA levels detected in qRT- PCR assay were normalized to total RNA levels measured with RiboGreenIM(ThennoFisher Scientific, Waltham, MA, USA).

[0372] Table 3: Sequences of Primer Probe Set for human INHBE

[0373] The results indicate that for the siRNA compounds assessed, ATsi967 and ATsi965 were more potent than the reference siRNA ATsi972.

[0374] In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in HPH Cells

[0375] Three siRNAs were selected from the above transfection study for further assessment in human primary hepatocytes (HPH) through free uptake. siRNAs were delivered at different final concentrations to HPH through free uptake, i.e., siRNAs are incubated with cells in the absence of transfection reagents and enter cells through endocytosis via GalNAc conjugate and ASGR receptor interactions. Cells were incubated for 72 hr and total RNA prepared using AcroPrep Advance 96- well Filter Plates (Pall Corporation, Port Washington, New York) and Qiagen’s RLT, RW1 and RPE buffers (Qiagen, Hilden, Germany). INHBE mRNA levels were determined through qRT-PCR using INHBE specific primer probe sets as listed in Table 3. qRT-PCR was performed using AgPath-lD™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The INHBE RNA levels detected in qRT-PCR assay were normalized to total RNA levels as measured with RiboGreenI M(ThermoFisher Scientific, Waltham, MA, USA). 'The results are shown in Figure 2. The IC50 values are calculated and shown in Table 5.

[0376] Tabic 5 IC50 of siRNA inhibition of INHBE mRNA in HPH

[0377] The results indicate that of the siRNA compounds assessed, ATsi963 had similar potency compared to the reference siRNA ATsi972 when delivered through free uptake. EXAMPLE 2: DESIGNING INHBE siRNAS WITH VARYING CHEMICAL MOTIFS

[0378] Additional ARNATAR siRNA compounds targeting INHBE were designed and assessed for activity in inhibiting INHBE mRNA levels. New siRNAs ATsi974-ATsi976 were based on previously disclosed ATsi965. New siRNA ATsi977 was based on previously disclosed ATsi968.

[0379] New siRNAs ATsi978-ATsi979 were based on previously disclosed ATsi967. The sequence and chemistry of the new siRNAs are disclosed in Table 6.

[0380] Table 6. INHBE siRNAs with Modifications to Both Strands

[0381] In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in Hep3B Cells siRNAs described in Table 6 were transfected at 0-10 nM into Hep3B cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 16 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 3. The IC50 of the siRNAs targeting INHBE are shown in Table 7, and the dose responses of siRNAs on INHBE mRNA levels in Hep3B cells are shown in Figure 3. INHBE mRNA levels were determined through qRT-PCR using INHBE specific primer probe sets as listed in Table 3. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The INHBE target RNA levels detected in qRT- PCR assay were normalized to total RNA levels measured with RiboGreen™ (ThermoFisher Scientific, Waltham, MA, USA).

[0382] Table 7: siRNA Activity Targeting INHBE in Hep3B cells

[0383] The results indicate that for the siRNA compounds assessed, ATsi978, ATsi979 and ATsi967 were more potent than reference siRNA ATsi972. EXAMPLE 3: DESIGNING INHBE siRNAS WITH VARYING CHEMICAL MOTIFS

[0384] Additional ARNATAR siRNA compounds targeting INHBE were designed and assessed for activity in inhibiting INHBE mRNA levels. The sequence and chemistry of the new siRNAs are disclosed in Table 8.

[0385] Table 8. INHBE siRNAs with Modifications to Both Strands

[0386] In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in Hep3B Ceils siRNAs described in Table 8 were transfected at 0-10 nM into Hep3B cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 16 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 3. The IC50 of the siRNAs targeting INHBE are shown in Table 9 and the dose responses of siRNAs on INHBE mRNA levels in Hep3B cells are shown in Figure 4. INHBE mRNA levels were determined through qRT-PCR using INHBE specific primer probe sets as listed in Table 3. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The INHBE target RNA levels detected in qRT- PCR assay were normalized to total RNA levels measured with RiboGreen™ (ThermoFisher

[0387] Scientific, Waltham, MA, USA).

[0388] Table 9: siRNA Activity Targeting INHBE in Hep3B cells

[0389] Several siRNA compounds assessed (ATsi980, ATsi978 and ATsi967) showed greater activity in inhibiting INHBE mRNA compared to the reference siRNA ATsi972.

[0390] EXAMPLE 4; DESIGNING INHBE siRNAS WITH VARYING CHEMICAL MOTIFS BUT WITHOUT A GALNAC CONJUGATE

[0391] Additional ARNATAR siRNA compounds targeting INHBE were designed and assessed for activity in inhibiting INHBE mRNA levels. The sequence and chemistry of the new siRNAs are disclosed in Table 10. The newly designed siRNAs were not conjugated to an N- Acetylgalactosamine-comprising moiety (GalNAc).

[0392] Table 10. INHBE siRNAs with Modifications to Both Strands

[0393] In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in HepSB Cells

[0394] The unconjugated siRNAs described in Table 10 were transfected at 0- 10 nM into Hep3B ceils with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 3. The IC50 of the siRNAs targeting INHBE are shown in Table 11 and the dose responses of siRNAs on INHBE mRNA levels in Hep3B cells are shown in Figure 5. INHBE mRNA levels were determined through qRT-PCR using INHBE specific primer probe sets as listed in Table 3. qRT- PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The INHBE target RNA levels detected in qRT-PCR assay were normalized to total RNA levels measured with RiboGreen™

[0395] (ThermoFisher Scientific, Waltham, MA, USA).

[0396] Table 11: siRNA Activity Targeting INHBE in Hep3B cells

[0397] The newly designed siRNA compounds without GalNAc conjugation showed similar or greater activity in inhibiting INHBE mRNA compared to the reference siRNA ATsi972.

[0398] EXAMPLE 5: DESIGNING INHBE siRNAS WITH VARYING CHEMICAL MOTIFS AND WITH A GALNAC CONJUGATE

[0399] Active siRNA compounds from Example 4 were conjugated with a GalNAc. The sequence and chemistry of the new conjugated siRNAs are disclosed in Table 12. The “old name” in Table 12 is the parental siRNA without GalNAc conjugation from the previous example.

[0400] Table 12. INHBE siRNAs with Modifications to Both Strands

[0401] In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in Hep3B Cells

[0402] The unconjugated siRNAs described in Table 12 were transfected at 0-10 nM into Hep3B cells with RNAiMAX (Invitrogen, Waltham, MA) and the cells further cultured for 24 hr. siRNA activity was determined by qRT-PCR using the primer probe sets as listed in Table 3. The IC50 of the siRNAs targeting INHBE are shown in Table 13 and the dose responses of siRNAs on INHBE mRNA levels in Hep3B cells are shown in Figure 6. INHBE mRNA levels were determined through qRT-PCR using INHBE specific primer probe sets as listed in Table 3. qRT- PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The INHBE target RNA levels detected in qRT-PCR assay were normalized to total RNA levels measured with RiboGreen™ (ThermoFisher Scientific, Waltham, MA, USA).

[0403] Table 13: siRNA Activity Targeting INHBE in Hep3B cells

[0404] The results show that the GalNAc conjugated siRNAs had similar or greater potency than reference siRNA ATsi972.

[0405] EXAMPLE 6: ASSESSING ARNATAR siRNAS TARGETING INHBE IN VIVO

[0406] To assess human INHBE siRNA activity in vivo, a mouse model expressing human 3xHA- INHBE-3xFLAG (also known as HA-INHBE-FLAG) fusion protein was made. This transgenic mouse model was chosen due to tlie lack of a human specific INHBE antibody so that an anti- FLAG antibody could be used to detect the HA-INHBE-FLAG fusion protein in the transgenic mouse liver. The human INHBE is tagged at its 5’ end with 3xHA peptide, and 3* end with 3xFLAG peptide. The double-tagged human INHBE protein is slightly larger than the endogenous mouse INHBE protein, enabling tlie detection of human INHBE using an anti-FLAG antibody.

[0407] Seven-week-old BALB / c mice were injected intravenously with adeno-associated virus (AAV) serotype AAV8 expressing human INHBE mRNA at a virus titer of 5.00E+11 (GenBank NM 031479.5; Sands MS, AAV-Mediated Liver-Directed Gene Therapy, 2014, Methods Mol Biol, 807:141-157). The AAV8+INHBE construct encoded an INHBE protein fused with an HA and FLAG tag (an octapeptide tag) at N- and C-terminus, respectively.

[0408] Two weeks after virus administration, five selected siRNAs (ATsi963, ATsi967, ATsi 1169, ATsi1170, ATsil 172) and reference siRNA ATsi972 were dosed subcutaneously at 5 mg / kg to the HA-INHBE-FLAG transgenic mice (N:::4-6 for each siRNA cohort). Four and half weeks after siRNA treatment, mice were sacrificed or biopsied, and liver proteins were prepared from the liver samples using RIPA Lysis and Extraction Buffer (ThermoFisher Scientific, Waltham, MA, USA). 600 μL RIPA Buffer (including Halt™ Protease and Phosphatase Inhibitor Cocktail (100X), ThennoFisher Scientific, Waltham, MA, USA Catalog #78440) were added to the liver samples and ground in Fisherbrand™ Pre-Filled Bead Mill Tubes (FisherScientific, Waltham, MA, USA, Catalog #15340153), 20 seconds on, 5 minutes on ice, repeat 3 times; then the liver lysate was centrifuged at 15000 rpm for 30 minutes. 2 mg total liver protein was used in immunoprecipitation assay to isolate HA-INHBE-FLAG protein.

[0409] Immunoprecipitation was performed as described below. Briefly, 20 μL. Anti-FLAG-M2- beads (Sigma-Aldrich, St. Louis, MO, USA; Catalog #M8823) were blocked with 5% milk in PBS overnight, and washed three times using RIP A Lysis and Extraction Buffer supplemented with ImM DTT. Cleaned beads were mixed with 2mg liver lysate from different mouse livers and incubated at 4°C for 2 hrs with rotation to allow the binding of HA-INHBE-FLAG on the beads. Next, the supernatant was removed after 1 minute setting on a magnetic rack. The beads were then washed 5 times using W100 buffer (50 mM Tris HC1 (pH 7.5), 100-mM KC1, 5-mM EDTA, 0.1 % NP-40, then transferred to clean tubes, and washed additional 3 times using W300 buffer (50-mM Tris-HCl (pH 7.5), 300-mM KC1, 5-mM EDTA, 0.1% NP-40). Wash buffer was then removed, and 50 μL 2X loading dye (ThermoFisher Scientific, Waltham, MA, USA Catalog #NP0007) was added to each tube. The beads were boiled for 7 mins, and 10 μL supernatant was loaded on SDS- PAGE, and the immunoprecipitated human HA-INHBE-FLAG fusion protein was detected using an anti-FLAG antibody (Abeam, Cambridge, UK; Catalog #ab! 162). The results are shown in Figures 7 and 9. The HA-INHBE-FLAG protein levels were quantified using Image J (an open source software for processing and analyzing scientific images), The average relative levels of human HA-INHBE-FLAG protein are shown in the charts in Figures 8 and 10.

[0410] The results showed that ARNATAR siRNAs ATsi1169 and ATsi1172 showed comparable activity in vivo compared with reference siRNA ATsi972 at the time point (4.5 weeks) evaluated.

[0411] EXAMPLE 7: DESIGN AND ASSESSMENT OF NEW siRNAS TARGETING INHBE

[0412] Active siRNA compounds from previous examples were redesigned with different chemical modifications and assessed for activity in inhibiting INHBE mRNA levels. The sequences and chemistries of the new siRNAs are disclosed in Table 14, along with the parent siRNAs from which they were derived in parentheses. Table 14: 1NHBE siRNAs with Chemical Modifications to Both Strands

[0413] In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in HPH Cells by Free Uptake

[0414] Five INHBE siRNAs were selected for assessment in human primary hepatocytes (HPH) through free uptake. A reference siRNA, ATsi972, was used as a positive control. siRNAs were delivered at different final concentrations to HPH cells through free uptake, i.e., siRNAs are incubated with cells in the absence of transfection reagents and enter cells through endocytosis via GalNAc conjugate and ASGR receptor interactions. The cells were incubated for 64 hr, and total RNA was prepared using AcroPrep Advance 96-well Filter Plates (Pall Corporation, Port Washington, New York) and Qiagen’s RLT, RW1, and RPE buffers (Qiagen, Hilden, Germany). INHBE mRNA levels were determined through qRT-PCR using INHBE- specific primer-probe sets as listed in Table 3. qRT-PCR was performed using AgPalh-ID™ One- Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The INHBE RNA levels detected in qRT-PCR assay were normalized to GAPDH mRNA levels. I’he results are shown in Figure 11. The IC50 values are calculated and shown in Table 15.

[0415] Table 15. IC50 of siRNA Inhibition of INHBE mRNA in HPH Cells

[0416] The results indicate that of the siRNA compounds assessed, ATsi1 l73, ATsi1226, and ATsi1227 had better potencies than the reference siRNA ATsi972 when delivered through free uptake to human primary hepatocytes. In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in Cells by Transfection

[0417] Six INHBE siRNAs were selected for assessment in Hep.3B cells through transfection. A reference siRNA, ATsi972, was used as a positive control.

[0418] Hep3B Cells (ATCC) were grown to approximately 60-80% confluency before varying doses of siRNA (0 nM, 0.016 nM, 0.08 nM, 0.4 nM, 2 nM and 10 nM) were transfected into cells using RNAiMAX (InVitrogen, Waltham, MA) according to the manufacturer’s recommended protocol, and the cells were further cultured for 24 hours. Total RNA was prepared from the cells using AcroPrep Advance 96-well Filter Plates (Pall Corporation, Port Washington, New York) using Qiagen's RLT, RW1, and RPE buffers (Qiagen, Hilden, Germany). RNA extraction procedures are done according to the manufacturer’s recommended protocols. siRNA activity was determined by measuring the levels of target mRNA through qRT-PCR using the INHBE primer- probe sets listed in Table 3. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA. USA). The target RNA levels detected in qRT-PCR assay were normalized to total RNA levels measured with Ribo Green™ (ThermoFisher Scientific, Waltham, MA, USA) in the aliquots of RNA samples used in qRT-PCR. The relative mRNA levels are shown in Figure 12. The ICsos were calculated and shown in Table 16.

[0419] Table 16. 1C50 of siRNA Inhibition of INHBE mRNA in Hep3b

[0420] The results indicate that several INHBE siRNA compounds assessed, ATsi1226, ATsi1253, ATsi1254, and ATsi1255, had better inhibitory activity than reference siRNA ATsi972 at 24 hours after transfection into Hep3b cells. In Vitro Assessment of siRNAs Targeting Human INHBE mRNA in HPH Cells by Free Uptake

[0421] The above INHBE siRNAs assessed by transfection in Hep3b cells were further evaluated in human primary' hepatocytes (HPH) through free uptake at different doses. siRNAs were delivered at different final concentrations to HPH cells through free uptake, i.e., siRNAs are incubated with cells in the absence of transfection reagents and enter cells through endocytosis via GalNAc conjugate and ASGR receptor interactions. After delivering the siRNAs to the cells, the cells were incubated for 64 hr, and total RNA was prepared using AcroPrep Advance 96-well Filter Plates (Pall Corporation, Port Washington, New York) and Qiagen’s RLT, RW1, and RPE buffers (Qiagen, Hilden, Germany). INHBE mRNA levels were determined through qRT-PCR using INHBE-specific primer-probe sets as listed in Table 3. qRT-PCR was performed using AgPath-ID™ One-Step RT-PCR Reagents in QS3 real-time PCR system ( ThermoFisher Scientific, Waltham, MA, USA). The INHBE RNA levels detected in qRT-PCR assay were normalized to GAPDH mRNA levels. The results are shown in Figure 13. The IC50 values are calculated and shown in Table 17.

[0422] Table 17. IC50 of siRNA Inhibition of INHBE mRNA in HPH Cells

[0423] The results from HPH study indicate that several INH BE siRNA compounds assessed, ATsi1226, ATsi1227, ATsi1253, ATsi1254, and ATsi1255, had similar or better inhibitory activity than the reference siRNA ATsi972 when delivered through free uptake to human primary hepatocytes. EXAMPLE 8: ASSESSING ARNATAR siRN'AS TARGETING INHBE IN VIVO

[0424] To assess human INHBE siRNA activity in vivo, a mouse model expressing human 3xHA- INHBE-3xFLAG (also known as HA-INHBE-FLAG) fusion protein was made, as described in Example 6, supra. Seven (7) to eight (8) week-old BALB / c mice were injected intravenously with adeno-associated virus (AAV) serotype AAV8 expressing human INHBE mRNA at a virus titer of5.00E+ll.

[0425] Fourteen (14) days after virus administration, two selected siRNAs (ATsi1255, ATsi1256) and reference siRNA ATsi972 were dosed subcutaneously at 5 mg / kg to the HA-INHBE-FLAG transgenic mice (N:::4 for each siRNA cohort). Saline was dosed as a control. Mice were biopsied at different times, 10 days (DIO), 21 days (D21), 35 days (D35), and sacrificed at day 57. Liver proteins were prepared from the liver samples using RIPA Lysis and Extraction Buffer (ThermoFisher Scientific, Waltham, MA, USA). 600 μL RIPA Buffer (including Halt™ Protease and Phosphatase Inhibitor Cocktail (100X), ThermoFisher Scientific, Waltham, MA, USA Catalog #78440) were added to the liver samples and ground in Fisherbrand™ Pre-Filled Bead Mill Tubes (FisherScientific, Waltham, MA, USA, Catalog #15340153), 20 seconds on, 5 minutes on ice, repeat 3 times; then the liver lysate was centrifuged at 15000 rpm for 30 minutes. 500 μg total liver protein was used in immunoprecipitation assay to isolate HA-INHBE-FLAG protein.

[0426] Immunoprecipitation was performed as described previously. Briefly, 20 ml Anti-FLAG- M2-beads (Sigma-Aldrich, St. Louis, MO, USA; Catalog #M8823) were blocked with 5% milk in PBS overnight, and washed three times using RIPA Lysis and Extraction Buffer supplemented with ImM DTT. Cleaned beads were mixed with 2mg liver lysate from different mouse livers, and incubated at 4’C for 2 hrs with rotation to allow the binding of HA-INHBE-FLAG on the beads. Next, the supernatant was removed after 1 min setting on the magnetic rack. The beads were then washed 5 times using W100 buffer (50 mM Tris-HCl (pH 7.5), 100-mM KC1, 5-mM EDTA, 0.1 % NP-40, then transferred to clean tubes, and washed additional 3 times using W300 buffer (50-mM Tris-HCl (pH 7.5), 300-mM KC1, 5-mM EDTA, 0.1% NP-40). Wash buffer was then removed, and 50 μL 2X loading dye (ThermoFisher Scientific, Waltham, MA, USA Catalog #NP0007 ) was added to each tube. The beads were boiled for 7 mins, and 10 μL. supernatant was loaded on SDS- PAGE, and the immunoprecipitated human HA-INHBE-FLAG fusion protein was detected using an anti-FLAG antibody (Abeam, Cambridge, UK; Catalog #abl 162). The results are shown in Figure 14. The HA-INHBE-FLAG protein levels were quantified using Image J (an open- source software for processing and analyzing scientific images). The average relative levels of human HA-INHBE-FLAG protein, relative to the level in the saline control animals, are shown in Table 18.

[0427] Table 18. Human INHBE Protein Level Over Tune in Mice

[0428] The results show that ARNATAR siRNAs ATsi1255 and ATsi1256 exhibited slightly better inhibitory activity in vivo compared with reference siRNA ATsi972 at time points D10 and D21. Moreover, the data at D35 and D57 indicate that ARNATAR siRNAs ATsi1255 and ATsi1256 can maintain a much longer duration of inhibition than control siRNA ATsi972 in the liver of transgenic mice.

[0429] EXAMPLE 9: ASSESSING ARNATAR siRNAS TARGETING INHBE IN VIVO

[0430] Similar to Example 8, seven to eight week-old BALB / c mice were injected intravenously with adeno-associated virus (AAV) serotype AAV8 expressing human INHBE rnRNA at a virus titer of 5.00E+11. The AAV8+INHBE construct encoded an INHBE protein fused with a 3xHA and 3xFLAG tag at N- and C-terminus, respectively.

[0431] Fourteen (14) days after virus administration, two selected siRNAs (ATsi1226, ATsi1255) and reference siRNA ATsi972 were dosed subcutaneously at 1 mg / kg, 3 mg / kg, and 9 mg / kg to the HA-INHBE-FLAG transgenic mice (N=3 for each siRNA cohort). Saline was dosed to a control cohort. Mice were biopsied at different times, 12 days (D12), 22 days (D22), 32 days (1)32), and sacrificed at day 47. Liver proteins were prepared from the liver samples using RIP A Lysis and Extraction Buffer (ThermoFisher Scientific, Waltham, MA, USA). 600 μL R1PA Buffer (including Halt™ Protease and Phosphatase Inhibitor Cocktail (100X), ThermoFisher Scientific, Waltham, MA, USA Catalog #78440) were added to the liver samples and ground in Fisherbrand™ Pre-Filled Bead Mill Tubes (FisherScientific, Waltham, MA, USA, Catalog #15340153), 20 seconds on, 5 minutes on ice, repeat 3 times; then the liver lysate was centrifuged at 15000 rpm for 30 minutes. 500 μg total liver protein was used in immunoprecipitation assay to isolate HA-INHBE-FLAG protein.

[0432] Immunoprecipitation was performed as described previously. The results for the 3mg / kg dosed group are shown in Figure 15. The HA-INHBE-FLAG protein levels were quantified using Image J (an open source software for processing and analyzing scientific images), The average relative levels of human HA-INHBE-FLAG protein for the 3mg / kg dosed group, relative to the level in the saline control animals, are shown in Table 19.

[0433] Tabic 19. Human INHBE Protein Level Over Time

[0434] The results show that ARNATAR siRNA ATsi1255 showed comparable activity at DI 2 with reference siRNA ATsi972, better activity at the time points D22, D34 and D47; Moreover, ARNATAR siRNA ATsil255 is demonstrated that it can maintain a much longer duration of inhibitory activity (see D34 and D47) than reference siRNA ATsi972 in the liver of transgenic mice.

[0435] Additionally, the half-maximal effective concentrations (EC50s), were calculated for siRNA ATsi1255 and reference siRNA ATsi972. The results are shown in Table 20. At Day 12, similar EC50s were observed for ATsi972 and ATsil255. However, at later days D22, D34, and D47, Amatar siRNA ATsi1255 showed much lower lC50s (0.584, 1.174, and 1.778), which indicates it has much better potency than reference siRNA ATsi972 (1.777, 3.078, and 1419) at those time points, This study shows that siRNA ATsi1255 has better activity and duration compared to the reference siRNA ATsi972, and that lower doses of ATsi1255 can be utilized compared to ATsi972.

[0436] Table 20. The Potencies (EC50s) of Different siRNAs Over Time

[0437] Table 21. SEQUENCE MASTER LIST

[0438]

Claims

What is claimed:

1. A double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cell, wherein the dsRNA compound comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the antisense strand comprises or is any of the antisense sequences in any one of Tables 2, 6, 8, 10, 12 or 14.

2. A double-stranded ribonucleic acid (dsRNA) compound for inhibiting expression of INHBE in a cel), wherein the dsRNA compound comprises a sense strand and an antisense strand forming the dsRNA compound, wherein the sense strand comprises any of the sense sequences in any one ofTables 2, 6, 8, 10, 12 or 14.

3. The double-stranded ribonucleic acid (dsRNA) compound of claim 1 or 2, wherein the dsRNA compound comprises the sense strand of claim 2 and the antisense strand of claim 1.

4. The double-stranded ribonucleic acid (dsRNA) compound of any preceding claim, wherein the dsRNA compound is an shRNA compound or an siRNA compound.

5. The double-stranded ribonucleic acid (dsRNA) compound of any preceding claim, wherein the dsRNA comprises at least one modified nucleotide.

6. The double-stranded ribonucleic acid (dsRNA) compound of any preceding claim, wherein the strand comprises at least one phosphorothioate intemucleotide (PS) linkage.

7. The double-stranded ribonucleic acid (dsRNA) compound of any of the preceding claims which is an siRNA comprising any one of siRNAs in any one of Tables 2, 6, 8, 10, 12 or 14.

8. The double-stranded ribonucleic acid (dsRNA) compound of any preceding claim, further comprising a conjugate.

9. The double-stranded ribonucleic acid (dsRNA) compound of claim 8, wherein the conjugate is an N-Acetylgalactosamine-comprising moiety (GalNAc).

10. The double-stranded ribonucleic acid (dsRNA) compound of any preceding claim, wherein the dsRNA compound inhibits expression of an INHBE target nucleic acid by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%.

11. A pharmaceutical composition for inhibiting expression of a gene encoding INHBE comprising the double-stranded ribonucleic acid (dsRNA) compound of any preceding claim, alone or in combination with a pharmaceutically acceptable carrier or excipient.

12. A method of treating and / or preventing an INHBE associated disease, disorder and / or condition in a subject, comprising administering to the subject a therapeutically effective amount of the dsRNA compound or pharmaceutical composition comprising the dsRNA compound of any preceding claim, thereby treating and / or preventing the INHBE associated disease, disorder and / or condition in the subject.

13. The method of claim 12, wherein the INHBE associated disease, disorder and / or condition is selected from the group consisting of metabolic, cardiovascular, hypertensive, inflammatory', cancerous, neurodegenerative, and / or hepatosteatotic diseases, disorders and / or conditions.

14. The method of claim 12, wherein the hepatosteatotic disease is nonalcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

15. The method of any one of claims 12-14, wherein the subject is an animal, preferably a human.

16. The method of any one of claims 12-15, further comprising administering to the subject an additional therapeutic agent for treatment of an INHBE associated disease, disorder and / or condition.

17. A kit comprising the dsRNA compound of any one of claims 1-10, or the pharmaceutical composition of claim 11 , and optionally, a label.

18. A process for preparing the sense and / or antisense strand of the double-stranded ribonucleic acid (dsRNA) compound of any one of claims 1-10, wherein the process comprises the steps of: a. preparing the sense and / or antisense strand by sequential coupling of modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis on a solid support; b. optionally, coupling an N-Acetylgalactosamine-comprising moiety (GalNAc) to the sense and / or antisense strand on the solid support via the phosphoramidite oligonucleotide synthesis; c. detaching the sense and / or antisense strand from the solid support and removing the solid support; and d. optionally, further purifying the sense and / or antisense strand, optionally using chromatography.

19. A process for preparing the sense and / or antisense strand of the double-stranded ribonucleic acid (dsRNA) compound of any one of claims 1-10, wherein the process comprises the steps of: a. coupling an N-Acctylgalactosamine-comprising moiety (GalNAc) to a solid support via the phosphoramidite oligonucleotide synthesis. b. coupling a modified and / or unmodified nucleotide via the phosphoramidite oligonucleotide synthesis to the GalNAc on the solid support; c. sequentially coupling additional modified and / or unmodified nucleotides via the phosphoramidite oligonucleotide synthesis to prepare the sense and / or antisense strand; d. detaching the sense and / or antisense strand from the solid support and removing the solid support; and e. optionally, further purifying the sense and / or antisense strand, optionally using chromatography.

20. A process of preparing the double-stranded ribonucleic acid (dsRNA) compound of any one of claims 1-10, comprising: a) contacting the sense strand prepared according to claim 41 or 42 with the antisense strand prepared according to claim 19 or 20 in equimolar concentrations in a solution; b) optionally heating the solution to a temperature of about 94°C; and c) optionally reducing the temperature of the solution to about 25°C.

21. The double-stranded ribonucleic acid (dsRNA) compound of claim 1 or 2, wherein the dsRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand comprises SEQ ID NO: 75 as shown in formula:, and the antisense strand comprises SEQ ID NO: 74 as shown in formula:

22. The double-stranded ribonucleic acid (dsRNA) compound of claim 1 or 2, wherein the dsRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand comprises SEQ ID NO: 77 as shown in formula:, and the antisense strand comprises SEQ ID NO: 76 as shown in formula:

23. The double-stranded ribonucleic acid (dsRNA) compound of claim I or 2, wherein the dsRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand comprises SEQ ID NO: 75 as shown in formula:, and the antisense strand comprises SEQ ID NO: 80 as shown in formula:

24. The double-stranded ribonucleic acid (dsRNA) compound of claim 1 or 2, wherein the dsRNA compound comprises a sense strand and an antisense strand forming a duplex, wherein the sense strand comprises SEQ ID NO: 77 as shown in formula:, and the antisense strand comprises SEQ ID NO: 81 as shown in formula:

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