Rnai constructs and methods for inhibiting expression of inhbe

Abstract

The disclosure relates to RNAi constructs, such as siRNA, for reducing expression of the INHBE gene. Methods of using such RNAi constructs to treat or prevent visceral obesity and diseases associated with visceral obesity, such as cardiovascular disease, are also described.

Description

RNAI CONSTRUCTS AND METHODS FOR INHIBITING EXPRESSION OF INHBEFIELD

[0001] The present disclosure relates to compositions and methods for modulating the expression of inhibin subunit beta E (INHBE) in the liver. In particular, the present disclosure relates to nucleic acid-based therapeutics for reducing INHBE expression via RNA interference and methods of using such nucleic acid-based therapeutics to treat diseases associated with increased visceral adipose tissue, such as cardiovascular disease.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: one 6.84 MB .xml file entitled “10694- US01-PRI_SequenceListing-Corrected,” created on November 5, 2024.BACKGROUND

[0003] Abdominal obesity, characterized by an accumulation of visceral adipose tissue, is a key risk factor for cardiovascular disease. Visceral adipose tissue or “VAT” is found beneath the abdominal wall in the spaces surrounding the liver, intestines, in an apron of tissue called the omentum, and other organs. In contrast, subcutaneous adipose tissue or “SAT” is found between the skin and the outer abdominal wall, accounting for 80-90% of total human adipose tissue (Sakers et al. Cell, 185: 419-446 (2022) and Karastergiou and Fried. Adv. Exp. Med. Biol. 1043: 29-51 (2017)). Traditional measures of obesity like body mass index (BMI) do not account for differences in fat distribution within the body and are poor predictors of risk of major adverse cardiovascular events (Yusuf et al., The Lancet, 366: 1640-1649 (2005)). Anthropomorphic measurements of waist and hip circumferences are better predictors of cardiovascular disease (CVD) risk because they serve as biomarkers for visceral adipose tissue and subcutaneous adipose tissue, respectively (Ross et al., Nature Reviews Endocrinology 16: 177-189 (2020); and Stefan, Lancet Diabetes Endocrinol., 8: 616-627 (2020)). In addition, genomic analyses including Mendelian randomization have demonstrated that increased distribution of visceral fat relative to subcutaneous fat is not simply abiomarker for cardiovascular disease, but rather is a causal risk factor (Emdin et al., JAMA, 317: 626-634 (2017); Shungin et al., Nature, 518: 187-196 (2015)). Consequently, there is an urgent unmet medical need to develop pharmacological agents against high-risk abdominal obesity.

[0004] Advances in human genetics have elucidated high-quality targets against visceral obesity, including a pathway that converges on activin receptor-like kinase 7 (ALK7), a serine / threonine kinase type I receptor in the TGF-beta superfamily that is preferentially expressed by adipocytes (Bertolino et al., Proc Natl Acad Sci USA, 105: 7246-7251 (2008); Emdin et al., Diabetes, 68: 226- 234 (2019); Ibanez, C.F., FEBS J., 289(19): 5776-5797 (2022); Justice et al., Nature Genetics, 51 : 452-469 (2019); Koprulu et al., J Clin Endocrinol Metab., 107(4): 1065-1077 (2022); Yogosawa et al., Diabetes, 62: 115-123 (2013); and Zhao et al., JCI Insight. 2023;8(4):el61229). Several molecules have been proposed to be putative ligands for ALK7. Activin E is a putative ligand for ALK7 comprised of a dimer of Inhibin subunit beta E subunits. Inhibin subunit beta E is encoded by the INHBE gene, which is highly expressed in the liver with little extra-hepatic expression. Loss-of- function variants in the gene encoding ALK7 (ACVR1C) or in INHBE are associated with reduced visceral adiposity and improved measures of cardiometabolic health, including dyslipidemia, reduced incidence of coronary artery disease, and risk of type II diabetes (Akbari et al., Nat Commun, 13: 4844 (2022); Deaton et al., Nature Comm., 13: 4319 (2022); Emdin et al., 2019, supra. Justice et al., supra, Shungin et al., supra). For example, whole-exome sequencing data from over 300,000 individuals identified rare loss of function variants in INHBE that were associated with lower waist-to-hip ratio adjusted for BMI (WHRadjBMI), which is a surrogate for abdominal adiposity (Deaton et al., supra). Thus, human genetics data supports the development of therapies that inhibit the ALK7 pathway.

[0005] There remains a need for agents and methods for inhibiting the ALK7 pathway to reduce visceral adiposity and abdominal obesity, ultimately leading to protection against cardiovascular disease.BRIEF SUMMARY

[0006] The present disclosure provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides of a sequence selected from the antisense sequences listed in Table 1 and Table 2,wherein the RNAi construct inhibits the expression of an inhibin subunit beta E (INHBE) mRNA sequence.

[0007] In some aspects, the sense strand of the RNAi construct comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length.

[0008] In some aspects, the duplex region of the RNAi construct is about 17 to about 24 base pairs in length. In some aspects, the duplex region of the RNAi construct is about 19 to about 21 base pairs in length.

[0009] In some aspects, the sense strand and the antisense strand of the RNAi construct are each independently about 19 to about 27 nucleotides in length. In some aspects, the sense strand and the antisense strand of the RNAi construct are each independently about 19 to about 23 nucleotides in length.

[0010] In some aspects, the RNAi construct comprises one or two blunt ends.

[0011] In some aspects, the RNAi construct comprises one or two nucleotide overhangs of 1 to 4 unpaired nucleotides. In some aspects, the nucleotide overhang comprises two unpaired nucleotides. In some aspects, the RNAi construct comprises a nucleotide overhang at the 3’ end of the sense strand, the 3’ end of the antisense strand, or the 3’ end of both the sense strand and the antisense strand. In some aspects, the nucleotide overhang comprises a 5’-UU-3’ dinucleotide or a 5’-dTdT- 3’ dinucleotide.

[0012] In some aspects, the RNAi construct comprises at least one modified nucleotide. In some aspects, the modified nucleotide is a 2’-modified nucleotide. In some aspects, the modified nucleotide is a 2’-fluoro modified nucleotide, a 2’-O-methyl modified nucleotide, a 2’-O- methoxy ethyl modified nucleotide, a 2’-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a deoxyribonucleotide, or combinations thereof. In some aspects, the modified nucleotide is a 2’-O- methyl modified nucleotide, a 2’-O-methoxyethyl modified nucleotide, a 2’-fluoro modified nucleotide, or combinations thereof. In some aspects, all of the nucleotides in the sense and antisense strands are modified nucleotides. In some aspects, the modified nucleotides are 2’-O- methyl modified nucleotides, 2’ -fluoro modified nucleotides, or combinations thereof.

[0013] In some aspects, the sense strand of the RNAi construct comprises an abasic nucleotide as the terminal nucleotide at its 3’ end, its 5’ end, or both its 3’ and 5’ ends. In some aspects, theabasic nucleotide is linked to an adjacent nucleotide through a 3 ’-3’ intemucleotide linkage or a 5’- 5’ internucleotide linkage.

[0014] In some aspects, the sense strand, the antisense strand, or both the sense and antisense strands of the RNAi construct comprise one or more phosphorothioate internucleotide linkages. In some aspects, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3’ end of the antisense strand. In some aspects, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3’ and 5’ ends of the antisense strand. In some aspects, the RNAi construct comprises at least one phosphorothioate internucleotide linkage at the 3’ end and / or the 5’ end of the sense strand. In some aspects, the sense strand comprises two consecutive phosphorothioate intemucleotide linkages between the terminal nucleotides at the 5’ end.

[0015] In some aspects, the antisense strand comprises or consists of a sequence selected from the antisense sequences listed in Table 1 and Table 2. In some aspects, the antisense strand comprises or consists of a sequence selected from SEQ NO: 642, SEQ ID NO: 660, SEQ ID NO: 681, SEQ ID NO: 701, SEQ ID NO: 1526, SEQ ID NO: 1544, SEQ ID NO: 1565, and SEQ ID NO: 1585.

[0016] In some aspects, the sense strand comprises or consists of a sequence selected from the sense sequences listed in Table 1 or Table 2. In some aspects, the sense strand comprises or consists of a sequence selected from SEQ ID NO: 200, SEQ ID NO: 218, SEQ ID NO: 239, SEQ ID NO: 259, SEQ ID NO: 1084, SEQ ID NO: 1102, SEQ ID NO: 1123, and SEQ ID NO: 1143.

[0017] In some aspects, the RNAi construct is any one of the duplex compounds listed in Table 1 or Table 2. For example, in some aspects, (a) the sense strand comprises or consists of the sequence of SEQ ID NO: 200 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 642; (b) the sense strand comprises or consists of the sequence of SEQ ID NO: 1084 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1526; (c) the sense strand comprises or consists of the sequence of SEQ ID NO: 218 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 660; (d) the sense strand comprises or consists of the sequence of SEQ ID NO: 1102 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1544; (e) the sense strand comprises or consists of the sequence of SEQ ID NO: 239 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 681; (f) the sensestrand comprises or consists of the sequence of SEQ ID NO: 1123 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1565; (g) the sense strand comprises or consists of the sequence of SEQ ID NO: 259 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 701; or (h) the sense strand comprises or consists of the sequence of SEQ ID NO: 1143 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1585.

[0018] In some aspects, the RNAi construct further comprises a ligand. In some aspects, the ligand binds to one or more proteins expressed on the surface of hepatocytes.

[0019] In some aspects, the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folate moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or antigen-binding fragment thereof. In some aspects, the ligand comprises galactose, galactosamine, or N-acetyl- galactosamine (GalNAc).

[0020] The disclosure also provides a composition including the RNAi construct and a pharmaceutically acceptable carrier, excipient, or diluent.

[0021] In some aspects, the disclosure provides a method for reducing the expression of INHBE in a patient in need thereof comprising administering to the patient the RNAi construct or the aforementioned composition. In other aspects, the disclosure provides a method for reducing visceral adiposity in a patient in need thereof comprising administering to the patient the RNAi construct or the composition. In some aspects, the patient is diagnosed with or at risk for cardiovascular disease. In other aspects, the disclosure provides a method for treating or preventing cardiovascular disease in a patient in need thereof comprising administering to the patient the RNAi construct or the composition. In some aspects, the cardiovascular disease is coronary artery disease, peripheral artery disease, myocardial infarction, or stroke.

[0022] In some aspects, the RNAi construct or composition is provided for reducing the expression of INHBE in a patient in need thereof, for use in a method of treating cardiovascular disease in a patient in need thereof, and / or for use in a method of reducing visceral adiposity in a patient in need thereof.

[0023] In some aspects, the disclosure provides an RNAi construct as described above or a composition comprising the RNAi construct for the preparation of a medicament for treating cardiovascular disease in a patient in need thereof.

[0024] The disclosure further provides the use of an RNAi construct as described above or a composition comprising the RNAi construct for reducing expression of INHBE in a patient in need thereof, and / or for reducing visceral adiposity in a patient in need thereof.

[0025] In some aspects, the expression level of INHBE in hepatocytes is reduced in the patient following administration of the RNAi construct as compared to the INHBE expression level in a patient not receiving the RNAi construct.

[0026] In some aspects, the patient has a visceral fat percentage of 10% or greater.DETAILED DESCRIPTION

[0027] The present disclosure is based, in part, on the design and generation of RNAi constructs that target the inhibin subunit beta E (INHBE) gene and reduce expression of INHBE in liver cells. In some embodiments, the gene may be within a cell or subject, such as a mammal (e.g., a human). In some embodiments, the disclosure provides compositions comprising RNAi constructs that target an INHBE mRNA and reduce INHBE expression in a cell or mammal. Such RNAi constructs and compositions are useful for reducing visceral adiposity in a subject, as well as treating or preventing metabolic and cardiovascular conditions associated with abdominal or visceral obesity, such as coronary artery disease, peripheral artery disease, myocardial infarction, and stroke.

[0028] INHBE, also referred to as inhibin (BE, is a growth factor that belongs to the transforming growth factor-0 (TGF-0) family. As discussed above, INHBE mRNA is predominantly expressed in the liver (Fang et al., Biochemical and Biophysical Res. Comm. 1996; 228(3):669-74; Hashimoto et al., Molecular and Cellular Endocrinology, 2002; 194(1-2): 117-22, and INHBE is involved in the regulation of liver cell growth and differentiation (Chabicovsky et al., Endocrinology. 2003; 144(8): 3497-504; and Vejda et al., Carcinogenesis. 2003; 24(11): 1801-9). INHBE protein dimerizes to form activin E, a hepatokine whose signaling is not well-characterized. It has been reported that insulin stimulates INHBE expression in liver cells. In addition, upregulation of INHBE mRNA has been observed in the livers of diet-induced obese mice, suggesting that INHBE is implicated in glucose metabolism (Hashimoto et al., Life sciences. 2009; 85(13- 14): 534-40). However, the association between insulin resistance and INHBE expression in the liver of humans has not been determined. Multiancestry exome sequencing studies suggest that inhibin 0E is a liver-expressed negative regulator of energy storage in peripheral adipose tissue in humans and that loss of itsfunction protects from liver inflammation, dyslipidemia, and type 2 diabetes by promoting healthy fat storage (Akbari et al., Nature Comm., 13: 4844 (2022)).

[0029] RNA interference (RNAi) is the process of introducing exogeneous RNA into a cell leading to specific degradation of the mRNA encoding the targeted protein with a resultant decrease in protein expression. Advances in both the RNAi technology and hepatic delivery, as well as growing positive outcomes with other RNAi-based therapies, suggest RNAi as a compelling means to therapeutically treat obesity and related cardiovascular conditions by directly targeting genes that regulate metabolism.

[0030] As used herein, the term “RNAi construct” refers to an agent comprising an RNA molecule that is capable of downregulating expression of a target gene (e.g., INHBE) via an RNA interference mechanism when introduced into a cell. “RNA interference” is the process by which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g. messenger RNA or mRNA molecule) in a sequence-specific manner, e.g. through an RNA induced silencing complex (RISC) pathway. In some embodiments, the RNAi construct comprises a doublestranded RNA (dsRNA) molecule comprising two antiparallel strands of contiguous nucleotides that are sufficiently complementary to each other to hybridize to form a duplex region. A doublestranded RNAi construct also may be referred to as an RNAi “trigger.” The terms “hybridize” or “hybridization” refer to the pairing of complementary polynucleotides, typically via hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary bases in the two polynucleotides. The strand comprising a region having a sequence that is substantially complementary to a target sequence (e.g., target mRNA) is referred to as the “antisense strand.” The “sense strand” refers to the strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may comprise a region that has a sequence that is substantially identical to the target sequence.

[0031] In certain embodiments, the sense strand and antisense strand of the double-stranded RNA may be two separate molecules that hybridize to form a duplex region but are otherwise unconnected. Such double-stranded RNA molecules formed from two separate strands are referred to as “small interfering RNAs” or “short interfering RNAs” (siRNAs). siRNAs are a class of noncoding, double-stranded RNA molecules that are typically about 20-27 base pairs and are central to RNAi. Thus, in some embodiments, the RNAi constructs of the disclosure comprise an siRNA. Inother embodiments, the RNAi construct may be a microRNA (also known as “miRNA” or “mature miRNA”). miRNAs are small (approximately 18-24 nucleotides in length), non-coding RNA molecules present in plants, animals, and some viruses. miRNAs resemble siRNA, but miRNAs originate from endogenous precursor hairpin RNA structures. miRNAs regulate gene expression by base-pairing to complementary regions of target mRNAs and directing the cleavage of the target RNA via the RISC pathway.

[0032] In some embodiments, the disclosure provides an RNAi construct directed to INHBE. In some embodiments, the RNAi construct is an siRNA that comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region that is complementary to an INHBE mRNA sequence. The region of the RNAi antisense strand may be complementary to any suitable region of a INHBE mRNA sequence. For example, the antisense strand may comprise a region that is complementary to the coding region or the 3’ untranslated region (UTR) of a INHBE mRNA sequence.

[0033] A double-stranded RNAi molecule may include chemical modifications to ribonucleotides, including modifications to the ribose sugar, base, or backbone components of the ribonucleotides, such as those described herein or known in the art. Any such modifications, as used in a double-stranded RNA molecule (e.g. siRNA, shRNA, or the like), are encompassed by the term “double-stranded RNA” for the purposes of this disclosure.

[0034] As used herein, a first sequence is “complementary” to a second sequence if a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a duplex region under certain conditions, such as physiological conditions. Other such conditions can include moderate or stringent hybridization conditions, which are known to those of skill in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if a polynucleotide comprising the first sequence base pairs with a polynucleotide comprising the second sequence over the entire length of one or both nucleotide sequences without any mismatches. A sequence is “substantially complementary” to a target sequence if the sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to a target sequence. Percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to bases at corresponding positions in a second or target sequence by the total length of the first sequence. A sequence may also be said tobe substantially complementary to another sequence if there are no more than 5, 4, 3, 2, or 1 mismatch over a 30 base pair duplex region when the two sequences are hybridized. Generally, if any nucleotide overhangs, as defined herein, are present, the sequence of such overhangs is not considered in determining the degree of complementarity between two sequences. By way of example, a sense strand of 21 nucleotides in length and an antisense strand of 21 nucleotides in length that hybridize to form a 19 base pair duplex region with a 2-nucleotide overhang at the 3’ end of each strand would be considered to be fully complementary as the term is used herein.

[0035] In some embodiments, a region of the antisense strand comprises a sequence that is fully complementary to a region of the target RNA sequence (e.g., INHBE mRNA). In such embodiments, the sense strand may comprise a sequence that is fully complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, e.g., having 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense and antisense strands. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g. within 6, 5, 4, 3, 2, or 1 nucleotides of the 5’ and / or 3’ ends of the strands). In one embodiment, any mismatches in the duplex region formed from the sense and antisense strands desirably occur within 6, 5, 4, 3, 2, or 1 nucleotides of the 5’ end of the antisense strand.

[0036] Where the two substantially complementary strands of a dsRNA are comprised of separate RNA molecules, those molecules need not, but can be, covalently connected. When the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs in the duplex is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

[0037] In other embodiments, the sense strand and the antisense strand that hybridize to form a duplex region may be part of a single RNA molecule, i.e., the sense and antisense strands are part of a self-complementary region of a single RNA molecule. In such cases, a single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3’ end of the sense strand is connected to the 5’ end of the antisense strand by a contiguous sequence of unpairednucleotides, which will form the loop region. The loop region is typically of a sufficient length to allow the RNA molecule to fold back on itself such that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region can comprise from about 3 to about 25, from about 5 to about 15, or from about 8 to about 12 unpaired nucleotides. As noted herein, such RNA molecules with at least partially self-complementary regions are referred to as “short hairpin RNAs” (shRNAs). In some embodiments, the loop region can comprise at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In other embodiments, the loop region can have 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer unpaired nucleotides. In certain embodiments, the RNAi constructs disclosed herein comprise an shRNA. The length of a single, at least partially self-complementary RNA molecule can be from about 35 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 85 nucleotides, or from about 50 to about 60 nucleotides and comprise a duplex region and loop region each having the lengths recited herein.

[0038] In some embodiments, the RNAi constructs disclosed herein comprise a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence that is substantially or fully complementary to a INHBE messenger RNA (mRNA) sequence. As used herein, a “INHBE mRNA sequence” refers to any messenger RNA sequence, including splice variants, encoding a INHBE protein, including INHBE protein variants or isoforms from any species (e.g. mouse, rat, non-human primate, human).

[0039] A INHBE mRNA sequence also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. A cDNA sequence refers to the sequence of an mRNA transcript expressed as DNA bases (e.g. guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g. guanine, adenine, uracil, and cytosine). Thus, the antisense strand of the RNAi constructs disclosed herein may comprise a region having a sequence that is substantially or fully complementary to a target INHBE mRNA sequence or INHBE cDNA sequence. A INHBE mRNA or cDNA sequence can include, but is not limited to, any INHBE mRNA or cDNA sequence such as can be derived from the Ensembl reference sequence ENST00000266646.3 or the NCBI reference sequences NM_031479.5 or XM_005571319.2.

[0040] A region of the antisense strand can be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the INHBE mRNA sequence. In some embodiments, the target region of the INHBE mRNA sequence to which the antisense strandcomprises a region of complementarity can range from about 15 to about 30 consecutive nucleotides, from about 16 to about 28 consecutive nucleotides, from about 18 to about 26 consecutive nucleotides, from about 17 to about 24 consecutive nucleotides, from about 19 to about 25 consecutive nucleotides, from about 19 to about 23 (e.g., 19, 20, 21, 22, or 23) consecutive nucleotides, or from about 19 to about 21 consecutive nucleotides. In certain embodiments, the region of the antisense strand comprising a sequence that is substantially or fully complementary to a INHBE mRNA sequence may, in some embodiments, comprise at least 19 contiguous nucleotides from an antisense sequence listed in Table 1. In some embodiments, the sense and / or antisense sequence comprises at least 15 consecutive nucleotides (e.g., at least 16, 17, 18, 19, 20, or 21 consecutive nucleotides) from a sequence listed in Table 1 with no more than 1, 2, or 3 nucleotide mismatches.

[0041] The sense strand of the RNAi construct typically comprises a sequence that is sufficiently complementary to the sequence of the antisense strand such that the two strands hybridize under physiological conditions to form a duplex region. A “duplex region” refers to the region in two complementary or substantially complementary polynucleotides that form base pairs with one another, either by Watson-Crick base pairing or other hydrogen bonding interaction, to create a duplex between the two polynucleotides. The duplex region of the RNAi construct should be of sufficient length to allow the RNAi construct to enter the RNA interference pathway, e.g., by engaging the Dicer enzyme and / or the RISC complex (described below). For instance, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths for the duplex region within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 27 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the duplex region is about 17 to about 24 base pairs in length. In another embodiment, the duplex region is about 19 to about 23 base pairs in length. For example, the duplex region may be about 19 base pairs or about 20 base pairs in length.

[0042] In some embodiments, an RNAi construct disclosed herein contains a duplex region of about 17 to about 24 nucleotides that interacts with a target RNA sequence, e.g., a INHBE targetmRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells can be broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III- like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3’ overhangs (Bernstein et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell, 107: 309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15: 188).

[0043] For embodiments in which the sense strand and antisense strand are two separate molecules (e.g., an siRNA RNAi construct), the sense strand and antisense strand need not be the same length as the length of the duplex region. For instance, one or both strands maybe longer than the duplex region and have one or more unpaired nucleotides or mismatches flanking the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that extend beyond the duplex region at the terminal ends of the strands. Nucleotide overhangs are typically created when the 3’ end of one strand extends beyond the 5’ end of the other strand or when the 5’ end of one strand extends beyond the 3’ end of the other strand. The length of a nucleotide overhang generally is between 1 and 6 nucleotides, 1 and 5 nucleotides, 1 and 4 nucleotides, 1 and 3 nucleotides, 2 and 6 nucleotides, 2 and 5 nucleotides, or 2 and 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some embodiments, the overhang comprises a 5’-uridine-uridine-3’ (5’-UU-3’) dinucleotide. In such embodiments, the UU dinucleotide may comprise ribonucleotides or modified nucleotides, e.g., 2’- modified nucleotides. In other embodiments, the overhang comprises a 5’-deoxythymidine- deoxythymidine-3’ (5’-dTdT-3’) dinucleotide.

[0044] The nucleotide overhang can be at the 5’ end or 3’ end of one or both strands. For example, in one embodiment, the RNAi construct comprises a nucleotide overhang at the 5’ end andthe 3’ end of the antisense strand. In another embodiment, the RNAi construct comprises a nucleotide overhang at the 5’ end and the 3’ end of the sense strand. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 5’ end of the sense strand and the 5’ end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3’ end of the sense strand and the 3’ end of the antisense strand, or the 3’ end of both the sense strand and the antisense strand.

[0045] The RNAi constructs may comprise a single nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other. A “blunt end” means that the sense strand and antisense strand are fully base-paired at the end of the molecule and there are no unpaired nucleotides that extend beyond the duplex region. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 3’ end of the sense strand and a blunt end at the 5’ end of the sense strand and 3’ end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3’ end of the antisense strand and a blunt end at the 5’ end of the antisense strand and the 3’ end of the sense strand. In certain embodiments, the RNAi construct comprises a blunt end at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and antisense strand have the same length and the duplex region is the same length as the sense and antisense strands (i.e., the molecule is double-stranded over its entire length).

[0046] The sense strand and antisense strand can each independently be any suitable length, such as about 15 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and antisense strand are each about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and antisense strand are of the same length but form a duplex region that is shorter than the strands such that the RNAi construct has two nucleotide overhangs. For instance, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand that are each 20 nucleotides in length, (ii) a duplex region that is 18 base pairs in length, and (iii) nucleotide overhangs of 2 unpaired nucleotides at both the 3’ end of the sense strand and the 3’ end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand that are each 23 nucleotides in length, (ii) a duplex region that is 21 basepairs in length, and (iii) nucleotide overhangs of 2 unpaired nucleotides at both the 3’ end of the sense strand and the 3’ end of the antisense strand. In other embodiments, the sense strand and antisense strand have the same length and form a duplex region over their entire length such that there are no nucleotide overhangs on either end of the double-stranded molecule. In one such embodiment, the RNAi construct is blunt ended and comprises (i) a sense strand and an antisense strand, each of which is 21 nucleotides in length, and (ii) a duplex region that is 21 base pairs in length. In another embodiment, the RNAi construct is blunt ended and comprises (i) a sense strand and an antisense strand, each of which is 23 nucleotides in length, and (ii) a duplex region that is 23 base pairs in length.

[0047] In other embodiments, the sense strand or the antisense strand is longer than the other strand and the two strands form a duplex region having a length equal to that of the shorter strand such that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand that is 20 nucleotides in length, (ii) an antisense strand that is 22 nucleotides in length, (iii) a duplex region of 20 base pairs in length, and (iv) a nucleotide overhang of 2 unpaired nucleotides at the 3’ end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand that is 20 nucleotides in length, (ii) an antisense strand that is 23 nucleotides in length, (iii) a duplex region of 20 base pairs in length, and (iv) a nucleotide overhang of 2 unpaired nucleotides at the 3’ end of the antisense strand and a nucleotide overhang of 1 unpaired nucleotide at the 5’ end of the antisense strand.

[0048] The antisense strand of the RNAi constructs of the disclosure can comprise or consist of the sequence of any one of the antisense sequences listed in Table 1 or Table 2, the sequence of nucleotides 1-21 or 1-23 of any of these antisense sequences, or the sequence of nucleotides 2-21 or 2-23 of any of these antisense sequences. Thus, in some embodiments, the antisense strand comprises or consists of a sequence selected from any one of SEQ ID NOs: 443-884 or SEQ ID NOs: 1327-1768. In other embodiments, the antisense strand comprises or consists of a sequence of nucleotides 1-21 of any one of SEQ ID NOs: 443-884 or SEQ ID NOs: 1327-1768. In still other embodiments, the antisense strand comprises or consists of a sequence of nucleotides 2-21 of any one of SEQ ID NOs: 443-884 or SEQ ID NOs: 1327-1768. In other embodiments, the antisense strand comprises or consists of a sequence of nucleotides 2-23 of any one of SEQ ID NOs: 443-884 or SEQ ID NOs: 1327-1768. In certain embodiments, the antisense strand comprises or consists of asequence selected from SEQ NO: 642, SEQ ID NO: 660, SEQ ID NO: 681, SEQ ID NO: 701, SEQ ID NO: 1526, SEQ ID NO: 1544, SEQ ID NO: 1565, and SEQ ID NO: 1585.

[0049] Likewise, the sense strand of the RNAi constructs disclosed herein can comprise or consist of the sequence of any one of the sense sequences listed in Table 1 or Table 2, the sequence of nucleotides 1-18, 1-19, or 1-20, of any of these sense sequences, or the sequence of nucleotides 2- 18, 2-19, or 2-20 of any of these sequences. Thus, in some embodiments, the sense strand comprises or consists of a sequence of nucleotides 1-18 of any one of SEQ ID NOs: 1-442 or SEQ ID NOs: 885-1326. In still other embodiments, the sense strand comprises or consists of a sequence of nucleotides 1-19 of any one of SEQ ID NOs: 1-442 or SEQ ID NOs: 885-1326. In further embodiments, the sense strand comprises or consists of a sequence of nucleotides 1-20 of any one of SEQ ID NOs: 1-442 or SEQ ID NOs: 885-1326. In other embodiments, the sense strand comprises or consists of a sequence of nucleotides 2-18 of any one of SEQ ID NOs: 1-442 or SEQ ID NOs: 885-1326. In some embodiments, the sense strand comprises or consists of a sequence of nucleotides 2-19 of any one of SEQ ID NOs: 1-442 or SEQ ID NOs: 885-1326. In yet other embodiments, the sense strand comprises or consists of a sequence of nucleotides 2-20 of any one of SEQ ID NOs: 1-442 or SEQ ID NOs: 885-1326. In certain embodiments, the sense strand comprises or consists of a sequence selected from SEQ ID NO: 200, SEQ ID NO: 218, SEQ ID NO: 239, SEQ ID NO: 259, SEQ ID NO: 1084, SEQ ID NO: 1102, SEQ ID NO: 1123, and SEQ ID NO: 1143.

[0050] In some embodiments, the RNAi construct of the present disclosure comprises (a) a sense strand comprising or consisting of the sequence of SEQ ID NO: 200 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 642; (b) a sense strand comprising or consisting of the sequence of SEQ ID NO: 1084 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 1526; (c) a sense strand comprising or consisting of the sequence of SEQ ID NO: 218 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 660; (d) a sense strand comprising or consisting of the sequence of SEQ ID NO: 1102 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 1544; (e) a sense strand comprising or consisting of the sequence of SEQ ID NO: 239 and an antisense strand comprises or consists of the sequence of SEQ ID NO: 681; (f) a sense strand comprising or consisting of the sequence of SEQ ID NO: 1123 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 1565; (g) a sense strand comprising or consisting of the sequence of SEQ ID NO: 259and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 701; or (h) a sense strand comprising or consisting of the sequence of SEQ ID NO: 1143 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 1585.

[0051] The RNAi construct of the disclosure can be any one of the duplex compounds listed in Table 1 or Table 2 (including the unmodified nucleotide sequences and / or modified nucleotide sequences of the compounds). In some embodiments, the RNAi construct is any of the duplex compounds listed in Table 1. In other embodiments, the RNAi construct is any of the duplex compounds listed in Table 2 (including the unmodified nucleotide sequences and / or modified nucleotide sequences of the compounds). In certain embodiments, the RNAi construct is duplex number 64848, 64847, 64846, or 64845 as listed in Table 1 or Table 2. In one particular embodiment, for example, the RNAi construct is duplex 64848. In another particular embodiment, the RNAi construct is duplex 64847. In another exemplary embodiment, the RNAi construct is duplex 64846. In another embodiment, the RNAi construct is duplex 64845.

[0052] The RNAi constructs disclosed herein, such as those listed in Table 1, may comprise one or more modified nucleotides. A “modified nucleotide” refers to a nucleotide that has one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not encompass ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate. However, the RNAi constructs may comprise combinations of modified nucleotides and ribonucleotides. Incorporation of modified nucleotides into one or both strands of double-stranded RNA molecules can improve the in vivo stability of the RNA molecules, e.g., by reducing the molecules’ susceptibility to nucleases and other degradation processes. The potency of RNAi constructs for reducing expression of the target gene can also be enhanced by incorporation of modified nucleotides.

[0053] In certain embodiments, the modified nucleotides have a modification of the ribose sugar. These sugar modifications can include modifications at the 2’ and / or 5’ position of the pentose ring as well as bicyclic sugar modifications. A 2’ -modified nucleotide refers to a nucleotide having a pentose ring with a substituent at the 2’ position other than OH. Such 2’ modifications include, but are not limited to, 2’-O-alkyl (e.g., O-C1-C10 or Q-C1-C10 substituted alkyl), 2’-O- allyl (O-CH2CH=CH2), 2’-C-allyl, 2’-fluoro, 2’-O-methyl (OCH3), 2’-O-methoxyethyl (O-(CH2)2OCH3), 2’-OCF3, 2’-O(CH2)2SCH3, 2’-O-aminoalkyl, 2’-amino (e.g., NH2), 2’-0-ethylamine, and 2’-azido. Modified nucleotides may also include deoxyribonucleotides, such as deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications at the 5’ position of the pentose ring include, but are not limited to, 5’-methyl (R or S); 5’-vinyl, and 5’-methoxy.

[0054] A “bicyclic sugar modification” refers to a modification of the pentose ring where a bridge connects two atoms of the ring to form a second ring resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification comprises a bridge between the 4’ and 2’ carbons of the pentose ring. Nucleotides comprising a sugar moiety with a bicyclic sugar modification are referred to herein as “bicyclic nucleic acids,” “bridged nucleic acids,” or “BNAs.” A “locked nucleic acid” (LNA) is a 2’,4’-bicyclic nucleic acid (2’,4’-BNA) in which the ribose ring is locked by a methylene bridge that connects 2’-oxygen and 4’-carbon. Exemplary bicyclic sugar modifications include, but are not limited to, a-L-Methyleneoxy (4’- CH2-O-2’) bicyclicnucleic acid (BNA); p-D -Methyleneoxy (4’-CH2-O-2’) BNA (LNA); Ethyleneoxy ( 4’-( CH2)2-O-2’) BNA;Aminooxy ( 4’- CH2-O-N(R)- 2’)BNA; Oxy amino (4’- CH2-N(R)-O-2’) BNA; Methyl(methyleneoxy) (4’-CH(CH3)-O-2’) BNA (also referred to as constrained ethyl or cEt); methylene-thio (4’- CH2-S-2’) BNA; methylene-amino (4’- CH2-N(R)-2’) BNA; methyl carbocyclic (4’- CH2-CH(CH3)-2’) BNA; propylene carbocyclic (4’-( CH2)3-2’) BNA; and Methoxy(ethyleneoxy) (4’-CH(CH2OMe)-O-2’)BNA (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the RNAi constructs disclosed herein are described in, e.g., U.S. Patent 9,181,551, U.S. Patent Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, 19: 937-954 (2012).

[0055] In some embodiments, the RNAi constructs comprise one or more 2’-fluoro modified nucleotides, 2’-O-methyl modified nucleotides, 2’-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2’-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In certain embodiments, the RNAi constructs comprise one or more 2’-fluoro modified nucleotides, 2’-O-methyl modified nucleotides, 2’-O-methoxyethyl modified nucleotides, or combinations thereof. In one particular embodiment, the RNAi constructs comprise one or more 2’ -fluoro modified nucleotides, 2’-O-methyl modified nucleotides, or combinations thereof.

[0056] Both the sense and antisense strands of the RNAi constructs can comprise one or multiple modified nucleotides. For instance, in some embodiments, the sense strand comprises 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides can be 2’-fluoro modified nucleotides, 2’-O-methyl modified nucleotides, or combinations thereof.

[0057] In certain embodiments, the modified nucleotides incorporated into one or both of the strands of the RNAi constructs of the disclosure have a modification of the nucleobase (also referred to herein as “base”). A “modified nucleobase” or “modified base” refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G) and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases can be synthetic or naturally occurring modifications and include, but are not limited to, universal bases, 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6- methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3 -deazaguanine and 3 -deazaadenine.

[0058] In some embodiments, the modified base is a universal base. A “universal base” refers to a base analog that indiscriminately forms base pairs with all of the natural bases in RNA and DNA without altering the double helical structure of the resulting duplex region. Universal bases are known to those of skill in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides, and nitroazole derivatives, such as 3- nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0059] Other suitable modified bases that can be incorporated into the RNAi constructs of the disclosure include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol. 10: 297- 310 (2000) and Peacock et al., J. Org. Chem., Vol. 76: 7295-7300 (2011) both of which are hereby incorporated by reference in their entireties. The skilled person is well aware that guanine, cytosine, adenine, thymine, and uracil may be replaced by other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of a polynucleotide comprising a nucleotide bearing such replacement nucleobase.

[0060] In some embodiments, the sense and / or antisense strands of the RNAi constructs may comprise one or more abasic nucleotides. An “abasic nucleotide” or “abasic nucleoside” is a nucleotide or nucleoside that lacks a nucleobase at the 1’ position of the ribose sugar. In certain embodiments, the abasic nucleotides are incorporated into the terminal ends of the sense and / or antisense strands of the RNAi constructs. In one embodiment, the sense strand comprises an abasic nucleotide as the terminal nucleotide at its 3’ end, its 5’ end, or both its 3’ and 5’ ends. In another embodiment, the antisense strand comprises an abasic nucleotide as the terminal nucleotide at its 3’ end, its 5’ end, or both its 3’ and 5’ ends. In such embodiments in which the abasic nucleotide is a terminal nucleotide, it may be an inverted nucleotide - that is, linked to the adjacent nucleotide through a 3 ’-3’ intemucleotide linkage (when on the 3’ end of a strand) or through a 5’ -5’ internucleotide linkage (when on the 5’ end of a strand) rather than the natural 3’-5’ internucleotide linkage. Abasic nucleotides may also comprise a sugar modification, such as any of the sugar modifications described above. In certain embodiments, abasic nucleotides comprise a 2’- modification, such as a 2’-fluoro modification, 2’-O-methyl modification, or a 2’-H (deoxy) modification. In one embodiment, the abasic nucleotide comprises a 2’-O-methyl modification. In another embodiment, the abasic nucleotide comprises a 2’-H modification (i.e. a deoxy abasic nucleotide).

[0061] In some embodiments, all pyrimidine nucleotides preceding an adenosine nucleotide in the sense strand and / or in the antisense strand are modified nucleotides. For example, where the sequence 5’-CA-3’ or 5’-UA-3’ appears in either strand, the cytidine and uridine nucleotides are modified nucleotides, preferably 2’-O-methyl modified nucleotides. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g. 2’-O-methyl modified nucleotides), and the 5’ nucleotide in all occurrences of the sequence 5’-CA-3’ or 5’-UA-3’ in theantisense strand are modified nucleotides (e.g. 2’-O-methyl modified nucleotides). In other embodiments, all nucleotides in the duplex region are modified nucleotides. In such embodiments, the modified nucleotides are preferably 2’-O-methyl modified nucleotides, 2’-fluoro modified nucleotides, or combinations thereof.

[0062] As discussed herein, when the RNAi construct comprises a nucleotide overhang, the nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides (e.g., 5’-uridine-uridine-3’ (5’-UU-3’) dinucleotide or 5’-deoxythymidine-deoxythymidine-3’ (5’- dTdT-3’) dinucleotide). For instance, in some embodiments, the nucleotides in the overhang are 2’- O-methyl modified nucleotides, 2’-fluoro modified nucleotides, 2 ’-methoxy ethyl modified nucleotides, or combinations thereof.

[0063] The RNAi constructs of the disclosure may also comprise one or more modified internucleotide linkages. As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage other than the natural 3’ to 5’ phosphodi ester linkage. In some embodiments, the modified intemucleotide linkage is a phosphorous-containing internucleotide linkage, such as a phosphotriester, an aminoalkyl phosphotri ester, an alkylphosphonate (e.g., methylphosphonate, 3’- alkylene phosphonate), a phosphinate, a phosphoramidate (e.g., 3’-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiralphosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, athionoalkylphosphotriester, and a boranophosphate. In one embodiment, a modified intemucleotide linkage is a 2’ to 5’ phosphodiester linkage. In other embodiments, the modified intemucleotide linkage is a non-phosphorous-containing intemucleotide linkage and thus can be referred to as a modified internucleoside linkage. Such non-phosphorous-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane linkages (-O-Si(H)2-O-); sulfide, sulfoxide and sulfone linkages; formacetyl and thioformacetyl linkages; alkene containing backbones; sulfamate backbones; methylenemethylimino (-CH2- N(CH3)-O-CH2-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S and CH2 component parts. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to create a peptide nucleic acid or PNA, such as those described in U.S. Patents 5,539,082; 5,714,331; and 5,719,262. Other suitable modified intemucleotide and internucleoside linkages that may beemployed in the disclosed RNAi constructs are described in U.S. Patents 6,693,187 and 9,181,551, U.S. Patent Publication No. 2016 / 0122761, and Deleavey and Damha, supra.

[0064] In certain embodiments, the RNAi constructs comprise one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages may be present in the sense strand, antisense strand, or both strands of the RNAi constructs. For instance, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate intemucleotide linkages. In other embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In still other embodiments, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate intemucleotide linkages. The RNAi constructs can comprise one or more phosphorothioate intemucleotide linkages at the 3 ’-end, the 5 ’-end, or both the 3’ - and 5’- ends of the sense strand, the antisense strand, or both strands. For instance, in certain embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate intemucleotide linkages at the 3 ’-end of the sense strand, the antisense strand, or both strands. In other embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate intemucleotide linkages at the 5’-end of the sense strand, the antisense strand, or both strands. In one embodiment, the RNAi construct comprises a single phosphorothioate intemucleotide linkage at the 3’ end of the sense strand and a single phosphorothioate intemucleotide linkage at the 3’ end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate intemucleotide linkages at the 3’ end of the antisense strand (i.e., a phosphorothioate intemucleotide linkage at the first and second intemucleotide linkages at the 3’ end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate intemucleotide linkages at both the 3’ and 5’ ends of the antisense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate intemucleotide linkages at both the 3’ and 5’ ends of the antisense strand and two consecutive phosphorothioate intemucleotide linkages at the 5’ end of the sense strand. In still another embodiment, the RNAi construct comprises two consecutive phosphorothioate intemucleotide linkages at both the 3’ and 5’ ends of the antisense strand and two consecutive phosphorothioate intemucleotide linkages at both the 3’ and 5’ ends of the sense strand (i.e. a phosphorothioate intemucleotide linkage at the first and second intemucleotide linkages at both the 5’ and 3’ ends of the antisense strand and aphosphorothioate intemucleotide linkage at the first and second internucleotide linkages at both the 5’ and 3’ ends of the sense strand). In any of the embodiments in which one or both strands comprise one or more phosphorothioate intemucleotide linkages, the remaining internucleotide linkages within the strands can be the natural 3’ to 5’ phosphodi ester linkages. For instance, in some embodiments, each intemucleotide linkage of the sense and antisense strands is selected from phosphodiester and phosphorothioate, wherein at least one intemucleotide linkage is a phosphorothioate.

[0065] In embodiments in which the RNAi construct comprises a nucleotide overhang, two or more of the unpaired nucleotides in the overhang can be connected by a phosphorothioate intemucleotide linkage. In certain embodiments, all the unpaired nucleotides in a nucleotide overhang at the 3’ end of the antisense strand and / or the sense strand are connected by phosphorothioate intemucleotide linkages. In other embodiments, all the unpaired nucleotides in a nucleotide overhang at the 5’ end of the antisense strand and / or the sense strand are connected by phosphorothioate intemucleotide linkages. In still other embodiments, all the unpaired nucleotides in any nucleotide overhang are connected by phosphorothioate intemucleotide linkages.

[0066] In some embodiments, the 5’ end of the sense strand, antisense strand, or both the antisense and sense strands of the disclosed RNAi constructs comprises a phosphate moiety. As used herein, the term “phosphate moiety” refers to a terminal phosphate group that includes unmodified phosphates (-0-P=0)(0H)0H) as well as modified phosphates. Modified phosphates include phosphates in which one or more of the O and OH groups are replaced with H, O, S, N(R) or alkyl where R is H, an amino protecting group or unsubstituted or substituted alkyl. Exemplary phosphate moi eties include, but are not limited to, 5 ’-monophosphate; 5 ’diphosphate; 5’- triphosphate; 5’-guanosine cap (7-methylated or non-methylated); 5’-adenosinecap or any other modified or unmodified nucleotide cap structure; 5 ’-monothiophosphate (phosphorothioate); 5’- monodithiophosphate (phosphorodi thioate); 5 ’-alpha-thiotriphosphate; 5 ’-gamma-thiotriphosphate, 5’-phosphoramidates; 5’-vinylphosphates; 5’-alkylphosphonates (wherein “alkyl” can be methyl, ethyl, isopropyl, propyl, etc.); and 5’-alkyletherphosphonates (wherein “alkylether” can be methoxymethyl, ethoxymethyl, etc ).

[0067] The modified nucleotides that can be incorporated into the RNAi constructs disclosed herein may have more than one chemical modification described herein. For instance, the modifiednucleotide may have a modification to the ribose sugar as well as a modification to the nucleobase. By way of example, a modified nucleotide may comprise a 2’ sugar modification (e.g., 2’ -fluoro or 2’-O-methyl) and comprise a modified base (e.g., 5-methyl cytosine or pseudouracil). In other embodiments, the modified nucleotide may comprise a sugar modification in combination with a modification to the 5’ phosphate that would create a modified intemucleotide or internucleoside linkage when the modified nucleotide is incorporated into a polynucleotide. For instance, in some embodiments, the modified nucleotide may comprise a sugar modification, such as a 2’ -fluoro modification, a 2’-O-methyl modification, or a bicyclic sugar modification, as well as a 5’ phosphorothioate group. Accordingly, in some embodiments, one or both strands of the RNAi constructs comprise a combination of 2’ modified nucleotides or BNAs and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of the RNAi constructs comprise a combination of 2’-fluoro modified nucleotides, 2’-O-methyl modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs comprising modified nucleotides and internucleotide linkages are shown in Table 2.

[0068] The RNAi constructs disclosed herein can readily be made using techniques known in the art, such as, for example, conventional nucleic acid solid phase synthesis. The polynucleotides of the RNAi constructs can be assembled on a suitable nucleic acid synthesizer utilizing standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers are sold commercially by several vendors, including DNA / RNA synthesizers from Applied Biosystems (Foster City, CA), MerMade synthesizers from BioAutomation (Irving, TX), and OligoPilot synthesizers from GE Healthcare Life Sciences (Pittsburgh, PA).

[0069] A 2’ silyl protecting group can be used in conjunction with acid labile dimethoxytrityl (DMT) at the 5’ position of ribonucleosides to synthesize oligonucleotides via phosphoramidite chemistry. Final deprotection conditions are known not to significantly degrade RNA products. All syntheses can be conducted in any automated or manual synthesizer on large, medium, or small scale. The syntheses may also be carried out in multiple well plates, columns, or glass slides.

[0070] The 2’ -O-silyl group can be removed via exposure to fluoride ions, which can include any source of fluoride ion, e.g., those salts containing fluoride ion paired with inorganic counterions, e.g., cesium fluoride and potassium fluoride or those salts containing fluoride ion paired with an organic counterion, e.g., a tetraalkylammonium fluoride. A crown ether catalyst can be utilized incombination with the inorganic fluoride in the deprotection reaction. Exemplary fluoride ion sources include, but are not limited to, tetrabutyl ammonium fluoride or aminohydrofluorides (e.g., combining aqueous HF with triethylamine in a dipolar aprotic solvent, e.g., dimethylformamide).

[0071] The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters towards fluoride. Methyl protection of the phosphotriester or phosphite triester can stabilize the linkage against fluoride ions and improve process yields.

[0072] Since ribonucleosides have a reactive 2’ hydroxyl substituent, it may be desirable to protect the reactive 2’ position in RNA with a protecting group that is orthogonal to a 5’-O- dimethoxytrityl protecting group, e.g., one stable to treatment with acid. Silyl protecting groups meet this criterion and can be readily removed in a final fluoride deprotection step that can result in minimal RNA degradation.

[0073] Tetrazole catalysts can be used in the standard phosphoramidite coupling reaction. Exemplary catalysts include, e.g., tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and pnitropheny Itetrazol e .

[0074] Additional methods of synthesizing the RNAi constructs described herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc., present on the bases) and protecting group methodologies (protection and deprotection) useful in synthesizing the RNAi constructs described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Custom synthesis of RNAi constructs is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).Ligands

[0075] The RNAi constructs disclosed herein may comprise a ligand. As used herein, a “ligand” refers to any compound or molecule that can interact with another compound or molecule, either directly or indirectly. The interaction of a ligand with another compound or molecule may elicit a biological response (e.g., initiate a signal transduction cascade, induce receptor mediated endocytosis) or may just be a physical association. The ligand can modify one or more properties of a double-stranded RNA molecule to which is attached, such as the pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties of the RNA molecule.

[0076] The ligand may comprise a serum protein (e.g., human serum albumin, low-density lipoprotein, globulin), a cholesterol moiety, a vitamin (e.g., biotin, vitamin E, vitamin Bl 2), a folate moiety, a steroid, a bile acid (e.g., cholic acid), a fatty acid (e.g., myristic acid (C14), palmitic acid (Cl 6), stearic acid (Cl 8), or docosanoic acid (C22)), a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), a glycoside, a phospholipid, or an antibody or binding fragment thereof (e.g., a whole antibody or binding fragment that targets the RNAi construct to a specific cell type, such as liver cells). Other examples of ligands include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, l,3-BisO(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, 03-(oleoyl)lithocholic acid, 03-( oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., antennapedia peptide, Tat peptide, RGD peptides), alkylating agents, polymers (e.g., polyethylene glycol (PEG ), PEG-40K), poly amino acids, and polyamines (e.g., spermine, spermidine).

[0077] In certain embodiments, the ligands have endosomolytic properties. Endosomolytic ligands promote the lysis of the endosome and / or transport of the RNAi construct, or its components, from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a poly cationic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of theendosome and / or transport of the RNAi construct, or its components, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, Vol. 26: 2964-2972 (1987)), the EALA peptide (Vogel et al., J. Am. Chem. Soc., Vol. 118: 1581-1586 (1996)), and their derivatives (Turk et al., Biochem. Biophys. Acta, Vol. 1559: 56- 68 (2002)). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0078] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroid. Cholesterol conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228 (2002)). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules have also been described in U.S. Patents 7,851,615; 7,745,608; and 7,833,992. In another embodiment, the ligand may comprise a folate moiety. Polynucleotides conjugated to folate moieties can be taken up by cells via a receptor-mediated endocytosis pathway. Such folate-polynucleotide conjugates are described in, e.g., U.S. Patent 8,188,247.

[0079] Given a lNHBE is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to specifically deliver the RNAi construct to liver cells. In some embodiments, RNAi constructs can be specifically targeted to the liver by employing ligands that bind to or interact with proteins expressed on the surface of liver cells. For example, in certain embodiments, a ligand may comprise one or more antigen binding proteins (e.g. antibodies or binding fragments thereof (e.g. Fab, scFv)) that specifically bind to a receptor expressed on hepatocytes.

[0080] In certain embodiments, the ligand comprises a carbohydrate. A “carbohydrate” refers to a compound made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched, or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, tri saccharides, tetrasaccharides, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides, such as starches, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from a pentose, hexose, or heptose and di- and tri-saccharides includingsuch monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N- acetylglucosamine.

[0081] In some embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In particular embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl- galactosamine (GalNAc) are particularly effective in targeting compounds to liver cells (see, e.g., D’Souza and Devarajan, J. Control Release, Vol. 203: 126-139 (2015)). Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs disclosed herein are described in U.S. Patents 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Publication No. 2003 / 0130186; and WIPO Publication No. WO 2013 / 166155.

[0082] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a “multivalent carbohydrate moiety” refers to a moiety comprising two or more carbohydrate units capable of independently binding or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains comprised of carbohydrates that can bind to two or more different molecules or two or more different sites on the same molecule. The valency of the carbohydrate moiety denotes the number of individual binding domains within the carbohydrate moiety. For instance, the terms “monovalent,” “bivalent,” “trivalent,” and “tetravalent” with reference to the carbohydrate moiety refer to carbohydrate moieties with one, two, three, and four binding domains, respectively. The multivalent carbohydrate moiety may comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety is bivalent, trivalent, or tetravalent. In such embodiments, the multivalentcarbohydrate moiety can be bi-antennary or tri-antennary. In one particular embodiment, the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the multivalent galactose moiety is trivalent or tetravalent. An exemplary GalNAc- containing ligand for incorporation into the RNAi constructs disclosed herein includes a tri- antennary GalNAc-containing ligand (also referred to as “GalNAc3”).

[0083] The ligand can be attached or conjugated to the RNA molecule of the RNAi construct directly or indirectly. For instance, in some embodiments, the ligand is covalently attached directly to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently attached via a linker to the sense or antisense strand of the RNAi construct. The ligand can be attached to nucleobases, sugar moi eties, or intemucleotide linkages of polynucleotides (e g., sense strand or antisense strand) of the RNAi constructs disclosed herein. Conjugation or attachment to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a ligand. Conjugation or attachment to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2, 5-, and 6-positions of a pyrimidine nucleobase can be attached to a ligand. Conjugation or attachment to sugar moieties of nucleotides can occur at any carbon atom. Exemplary carbon atoms of a sugar moiety that can be attached to a ligand include the 2’, 3’, and 5’ carbon atoms. The 1 ’ position also can be attached to a ligand, such as in abasic nucleotides. Internucleotide linkages can also support ligand attachments. For phosphorus-containing linkages (e g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like), the ligand can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the ligand can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0084] In certain embodiments, the ligand may be attached to the 3’ or 5’ end of either the sense or antisense strand. In certain embodiments, the ligand is covalently attached to the 5’ end of the sense strand. For example, in some embodiments, the ligand is attached to the 5 ’-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 5 ’-position of the 5’-terminal nucleotide of the sense strand. In other embodiments, the ligand is covalently attached to the 3’ end of the sense strand. For example, in some embodiments, the ligand is attachedto the 3 ’-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3’-position of the 3’-terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3’ end of the sense strand, but before one or more terminal nucleotides (i.e. before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2’ -position of the sugar of the 3 ’-terminal nucleotide of the sense strand.

[0085] In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. A “linker” is an atom or group of atoms that covalently joins a ligand to a polynucleotide component of the RNAi construct. The linker may be from about 1 to about 30 atoms in length, from about 2 to about 28 atoms in length, from about 3 to about 26 atoms in length, from about 4 to about 24 atoms in length, from about 6 to about 20 atoms in length, from about 7 to about 20 atoms in length, from about 8 to about 20 atoms in length, from about 8 to about 18 atoms in length, from about 10 to about 18 atoms in length, and from about 12 to about 18 atoms in length. In some embodiments, the linker may comprise a bifunctional linking moiety, which generally comprises an alkyl moiety with two functional groups. One of the functional groups is selected to bind to the compound of interest (e.g., sense or antisense strand of the RNAi construct) and the other is selected to bind essentially any selected group, such as a ligand as described herein. In certain embodiments, the linker comprises a chain structure or an oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups that are typically employed in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.

[0086] Linkers that may be used to attach a ligand to the sense or antisense strand in the RNAi constructs include, but are not limited to, pyrrolidine, 8-amino-3,6-di oxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate, 6-aminohexanoic acid, substituted Ci-Cio alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl. Preferred substituent groups for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0087] In certain embodiments, the linkers are cleavable. A cleavable linker is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release thetwo parts the linker is holding together. In some embodiments, the cleavable linker is cleaved at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0088] Cleavable linkers are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linker by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linker by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0089] A cleavable linker may comprise a moiety that is susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand inside the cell, or into the desired compartment of the cell.

[0090] A linker can include a cleavable group that is cleavable by a particular enzyme. The type of cleavable group incorporated into a linker can depend on the cell to be targeted. For example, liver-targeting ligands can be linked to RNA molecules through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other types of cells rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cells rich in peptidases, such as liver cells and synoviocytes.

[0091] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It will also be desirableto also test the candidate cleavable linker for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell- free or culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate linkers are cleaved at least 2, 4, 10, 20, 50, 70, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0092] In other embodiments, redox cleavable linkers are utilized. Redox cleavable linkers are cleaved upon reduction or oxidation. An example of reductively cleavable group is a disulfide linking group (-S-S-). To determine if a candidate cleavable linker is a suitable “reductively cleavable linker,” or, for example, is suitable for use with a particular RNAi construct and particular ligand, one or more methods described herein can be used. For example, a candidate linker can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent known in the art, which mimics the rate of cleavage that would be observed in a cell, e.g., a target cell. The candidate linkers can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a specific embodiment, candidate linkers are cleaved by at most 10% in the blood. In other embodiments, useful candidate linkers are degraded at least 2, 4, 10, 20, 50,70, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

[0093] In yet other embodiments, phosphate-based cleavable linkers are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that hydrolyzes phosphate groups in cells are enzymes, such as phosphatases. Examples of phosphate-based cleavable groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, - S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S- P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-, where Rk can be hydrogen or Ci- Cio alkyl. Specific embodiments include -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S- P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -SP(S)(OH)-O-, -O-P(O)(H)-O-,-O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. Another specific embodiment is -O-P(O)(OH)-O-. These candidate linkers can be evaluated using methods analogous to those described above.

[0094] In other embodiments, the linkers may comprise acid cleavable groups, which are groups that are cleaved under acidic conditions. In some embodiments, acid cleavable groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents, such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes, can provide a cleaving environment for acid cleavable groups. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A specific embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl, pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

[0095] In other embodiments, the linkers may comprise ester-based cleavable groups, which are cleaved by enzymes, such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable groups have the general formula -C(O)O-, or -OC(O) -. These candidate linkers can be evaluated using methods analogous to those described above.

[0096] In further embodiments, the linkers may comprise peptide-based cleavable groups, which are cleaved by enzymes, such as peptidases and proteases in cells. Peptide-based cleavable groups are peptide bonds formed between amino acids to yield oligopeptides (e g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (- C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula - NHCHRAC(O)NHCHRBC(O)-, where RAand RBare the side chains of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

[0097] Other types of linkers suitable for attaching ligands to the sense or antisense strands in the RNAi constructs described herein are known in the art and can include the linkers described in, e.g., U.S. Patents 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551.

[0098] In certain embodiments, the ligand covalently attached to the sense or antisense strand of the RNAi constructs comprises a GalNAc moiety, e.g., a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3’ end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5’ end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3’ end of the sense strand. In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5’ end of the sense strand. Multivalent GalNAc moieties that can be attached to the disclosed RNAi constructs are known in the art and can include GalNAc moieties described in, e.g., WO 2021 / 119034.Compositions and Methods

[0099] The disclosure also provides compositions and formulations comprising the RNAi constructs described herein and pharmaceutically acceptable carriers, excipients, or diluents. Such compositions and formulations are useful for reducing expression of INHBE in a subject in need thereof. Where clinical applications are contemplated, pharmaceutical compositions and formulations can be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0100] The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier, excipient, or diluent” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the RNAi constructs of thepresent disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or RNAi constructs of the compositions.

[0101] Compositions and methods for the formulation of pharmaceutical compositions depend on several criteria, including, but not limited to, route of administration, type and extent of disease or disorder to be treated, and dose to be administered. In some embodiments, the pharmaceutical compositions are formulated based on the intended route of delivery. For instance, in certain embodiments, the pharmaceutical compositions are formulated for parenteral delivery. Parenteral forms of delivery include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, and intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (e.g., GalNAc-containing ligands described herein).

[0102] In some embodiments, the pharmaceutical compositions comprise an effective amount of an RNAi construct described herein. An “effective amount” or “therapeutically effective amount” is an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, an effective amount is an amount sufficient to reduce INHBE gene expression in a particular tissue or cell type (e.g., liver or hepatocytes) of a subject. In some embodiments, an effective amount may be an amount sufficient to only partially reduce INHBE gene expression, for example, to a level comparable to expression of the wild-type INHBE allele in human heterozygotes. In some aspects, a pharmaceutical composition may comprise a prophylactically effective amount of the RNAi construct, so as to prevent at least one symptom in the subject having a disorder that would benefit from reduction in INHBE gene expression.

[0103] An effective amount of an RNAi construct disclosed herein may be from about 0.01 mg / kg body weight to about 100 mg / kg body weight. The pharmaceutical composition comprising an effective amount of RNAi construct can be administered weekly, biweekly, monthly, quarterly, or biannually. The precise determination of what would be considered an effective amount and frequency of administration may be based on several factors, including a patient’s size, age, gender, type of disorder to be treated (e.g., myocardial infarction, coronary artery disease, peripheral arterydisease, or stroke), particular RNAi construct employed, and route of administration. Estimates of effective dosages and in vivo half-lives for any particular RNAi construct disclosed herein can be ascertained by testing in appropriate animal models.

[0104] Administration of the pharmaceutical compositions of the present disclosure may be via any common route so long as the target tissue is available via that route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or by direct injection into liver tissue or delivery through the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For instance, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0105] Colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the RNAi constructs disclosed herein or vectors encoding such constructs. Commercially available fat emulsions that are suitable for delivering the nucleic acids include INTRALIPID®, LIPOSYN®, LIPOSYN®II, LIPOSYN®III, NUTRILIPID, and other similar lipid emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs may be encapsulated within liposomes, such as cationic liposomes. Alternatively, RNAi constructs disclosed herein may be complexed to lipids, such as cationic lipids. Suitable lipids and liposomes include neutral (e g., di oleoyl phosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), and dipalmitoyl phosphatidylcholine (DPPC)), distearolyphosphatidyl choline), negative (e.g., dimyristoylphosphatidyl glycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidyl ethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations also are disclosed in, e.g., U.S. Patents 5,783,565; 5,837,533; 5,981,505; 6,127,170; 6,217,900; 6,379,965; 6,383,512; 6,747,014; 7,202,227; and WO 03 / 093449.

[0106] In some embodiments, the RNAi constructs disclosed herein are fully encapsulated in a lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As usedherein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a noncationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are exceptionally useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent- nucleic acid complex as set forth in PCT Publication No. WO 00 / 03683. The nucleic acid-lipid particles typically have a mean diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids present in the nucleic acid-lipid particles desirably are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patents 5,976,567; 5,981,501; 6,534,484; 6,586,410; and 6,815,432; and PCT Publication No. WO 96 / 40964.

[0107] Pharmaceutical compositions suitable for injections include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the required particle size (in the case of dispersion), and / or by using surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents (e.g., sugars or sodium chloride) may be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including absorption-delaying agents, such as, for example, aluminum monostearate and gelatin.

[0108] Sterile injectable solutions may be prepared by incorporating an appropriate amount of the RNAi construct (alone or complexed with a ligand) into a solvent along with any otheringredients (such as described above) as desired, followed by fdtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0109] The compositions provided herein may be formulated in a neutral or salt form.Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric or phosphoric acids), or from organic acids (e g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, and the like).

[0110] For parenteral administration in an aqueous solution, for example, a solution generally is suitably buffered, and a liquid diluent is first rendered isotonic with, e.g., sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Sterile aqueous media desirably are employed as is known to those of skill in the art. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA standards. In certain embodiments of the disclosure, a pharmaceutical composition comprises or consists of a sterile saline solution and an RNAi construct described herein. In other embodiments, a pharmaceutical composition comprises or consists of an RNAi construct described herein and sterile water (e.g., water for injection, WFI). In still other embodiments, a pharmaceutical composition comprises or consists of an RNAi construct described herein and phosphate-buffered saline (PBS).[0U1] In some embodiments, the pharmaceutical compositions are packaged with or stored within a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, auto injectors, injection pumps, on-body injectors, and injection pens. Devices for aerosolized or powder formulations include, but are not limited to,inhalers, insufflators, aspirators, and the like. Thus, the present disclosure includes administration devices comprising a pharmaceutical composition for treating or preventing one or more of the disorders described herein.

[0112] In some embodiments, the RNAi constructs disclosed herein may be delivered to a cell or tissue of interest by administering a vector that encodes and controls the intracellular expression of the RNAi construct. A “vector” (also referred to herein as an “expression vector”) is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. A vector can be replicated in a living cell, or it can be made synthetically.

[0113] Generally, a vector for expressing an RNAi construct will comprise one or more promoters operably linked to sequences encoding the RNAi construct. The phrases “operably linked,” “operatively linked,” or “under transcriptional control” may be used interchangeably herein to indicate when a promoter is in the correct location and orientation in relation to a polynucleotide sequence to control the initiation of transcription by RNA polymerase and expression of the polynucleotide sequence. A “promoter” refers to a sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene sequence. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI or U6 RNA pol III, and viral promoters (e.g., human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In some embodiments, an HI or U6RNA pol III promoter is employed. The promoter can be a tissue-specific or inducible promoter. Of particular interest are liver-specific promoters, such as promoter sequences from the human alpha- 1 antitrypsin gene, albumin gene, hemopexin gene, and hepatic lipase gene. Inducible promoters include, for example, promoters regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-PDl -thiogalactopyranoside (IPTG).

[0114] When the RNAi construct comprises an siRNA, the two separate strands (sense and antisense strand) can be expressed from a single vector or two separate vectors. For example, in some embodiments, the sequence encoding the sense strand is operably linked to a promoter on afirst vector and the sequence encoding the antisense strand is operably linked to a promoter on a second vector. In such an embodiment, the first and second vectors are co-introduced, e.g., by infection or transfection, into a target cell, such that the sense and antisense strands, once transcribed, will hybridize intracellularly to form the siRNA molecule. In another embodiment, the sense and antisense strands are transcribed from two separate promoters located in a single vector. In such embodiments, the sequence encoding the sense strand may be operably linked to a first promoter and the sequence encoding the antisense strand may be operably linked to a second promoter, wherein the first and second promoters are located in a single vector. In one embodiment, the vector comprises a first promoter operably linked to a sequence encoding the siRNA molecule, and a second promoter operably linked to the same sequence in the opposite direction, such that transcription of the sequence from the first promoter results in the synthesis of the sense strand of the siRNA molecule and transcription of the sequence from the second promoter results in synthesis of the antisense strand of the siRNA molecule.

[0115] When the RNAi construct comprises a shRNA, a sequence encoding the single, at least partially self-complementary RNA molecule is operably linked to a promoter to produce a single transcript. In some embodiments, the sequence encoding the shRNA comprises an inverted repeat joined by a linker polynucleotide sequence to produce the stem and loop structure of the shRNA following transcription.

[0116] In some embodiments, the vector encoding an RNAi construct is a viral vector. Various viral vector systems that are suitable to express the RNAi constructs described herein include, but are not limited to, adenoviral vectors, retroviral vectors (e g., lentiviral vectors, maloney murine leukemia virus), adeno-associated viral vectors; herpes simplex viral vectors; SV40 vectors; polyoma viral vectors; papilloma viral vectors; picomaviral vectors; and pox viral vectors (e.g., vaccinia virus). In certain embodiments, the viral vector is a retroviral vector (e.g., lentiviral vector).

[0117] Various vectors suitable for use in the disclosure, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into vectors, and methods of delivering the vectors to the cells of interest are known in the art (see, e.g., Dornburg , Gene Therap., Vol. 2: 301-310 (1995); Eglitis, Biotechniques, Vol. 6: 608-614 (1988); Miller, HumGene Therap., Vol. 1 : 5-14 (1990); Anderson, Nature, Vol. 392: 25-30 (1998); Rubinson D A et al., Nat. Genet., Vol. 33: 401- 406 (2003); Brummelkamp et al., Science, Vol. 296: 550-553 (2002); Brummelkamp et al., CancerCell, Vol. 2: 243-247 (2002); Lee et al., Nat Biotechnol, Vol. 20: 500-505 (2002); Miyagishi et al., Nat Biotechnol, Vol. 20: 497-500 (2002); Paddison et al., GenesDev, Vol. 16: 948-958 (2002); Paul et al., Nat Biotechnol, Vol. 20: 505-508 (2002); Sui et al., Proc Natl Acad Sci USA, Vol. 99: 5515- 5520 (2002); and Yu et al., Proc Natl Acad Sci USA, Vol. 99: 6047-6052 (2002)).

[0118] The present disclosure also provides methods of inhibiting expression of an INHBE gene, and thus the production of INHBE protein, in a cell. The methods include contacting a cell with an any one of the RNAi constructs described herein in an amount effective to inhibit expression of INHBE in the cell. The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

[0119] The phrase “inhibiting expression of an INHBE gene” is intended to refer to inhibition of expression of any INHBE gene (such as, e.g., a mouse INHBE gene, a rat INHBE gene, a monkey INHBE gene, or a human INHBE gene) as well as variants or mutants of a INHBE gene. Thus, the INHBE gene may be a wild-type INHBE gene, a mutant INHBE gene, or a transgenic INHBE gene in the context of a genetically manipulated cell, group of cells, or organism.

[0120] “Inhibiting expression of a INHBE gene” includes any level of inhibition of a INHBE gene, e.g., at least partial suppression of the expression of a INHBE gene. The expression of the INHBE gene may be assessed based on the level, or the change in the level, of any variable associated with INHBE gene expression, e.g., INHBE mRNA level or INHBE protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample obtained from a subject.

[0121] INHBE expression can be assessed by measuring the amount or level of INHBE mRNA, INHBE protein, or another biomarker linked to INHBE expression. The reduction of INHBE expression in cells or animals treated with an RNAi construct disclosed herein can be determined relative to the INHBE expression in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For instance, in some embodiments, reduction of INHBE expression is assessed by (a) measuring the amount or level of INHBE mRNA in liver cells treated with a RNAi construct disclosed herein, (b) measuring the amount or level of INHBE mRNA in liver cells treated with a control RNAi construct (e.g., an RNAi construct directed to a RNA molecule not expressed in liver cells or a RNAi construct having a nonsense or scrambled sequence) or no construct, and (c)comparing the measured INHBE mRNA levels from treated cells in (a) to the measured INHBE mRNA levels from control cells in (b). The INHBE mRNA levels in the treated cells and controls cells can be normalized to RNA levels for a control gene (e.g., 18S ribosomal RNA) prior to comparison. INHBE mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reversetranscriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

[0122] In other embodiments, reduction of INHBE expression is assessed by (a) measuring the amount or level of INHBE protein in liver cells treated with a RNAi construct disclosed herein, (b) measuring the amount or level of INHBE protein in liver cells treated with a control RNAi construct (e g., RNAi construct directed to a RNA molecule not expressed in liver cells or a RNAi construct having a nonsense or scrambled sequence) or no construct, and (c) comparing the measured INHBE protein levels from treated cells in (a) to the measured INHBE protein levels from control cells in (b). INHBE protein levels can be measured using any suitable method known to those of skill in the art, including but not limited to, western blots, immunoassays (e.g., ELISA), and flow cytometry. Any suitable method of measuring INHBE mRNA or protein can be used to assess the efficacy of the RNAi constructs disclosed herein.

[0123] In some embodiments, the methods to assess INHBE expression levels are performed in vitro in cells that natively express INHBE (e.g., liver cells) or cells that have been engineered to express INHBE. In certain embodiments, the methods are performed in vitro in liver cells. Suitable liver cells include, but are not limited to, primary hepatocytes (e.g. human, non-human primate, or rodent hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the liver cells are Hep3B cells. In another embodiment, the liver cells are HepG2 cells.

[0124] In other embodiments, the methods to assess INHBE expression levels are performed in vivo. For example, the RNAi constructs and any control RNAi constructs can be administered to an animal (e.g., rodent or non-human primate), and INHBE mRNA or protein levels may be assessed in liver tissue harvested from the animal following treatment. Alternatively or additionally, a biomarker or functional phenotype associated with INHBE expression can be assessed in the treated animals.

[0125] In certain embodiments, expression of INHBE is reduced in liver cells by at least 40%, at least 45%, or at least 50% by an RNAi construct disclosed herein. In some embodiments, expression of INHBE is reduced in liver cells by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by an RNAi construct disclosed herein. In other embodiments, the expression of INHBE is reduced in liver cells by about 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by an RNAi construct disclosed herein. Reduction of INHBE can be measured using a variety of techniques including, for example, RNA FISH or droplet digital PCR (see, e.g., Kamitaki et al., Digital PCR. Methods in Molecular Biology, 1768'. 401-422 (2018). doi : 10.1007 / 978- 1 -4939-7778-9_23).

[0126] In some embodiments, an ICso value is calculated to assess the potency of an RNAi construct disclosed herein for inhibiting INHBE expression in liver cells. An “IC50 value” is the dose / concentration required to achieve 50% inhibition of a biological or biochemical function. In other embodiments, the potency of an RNAi construct may be assessed by calculating an “AC50” value, which is the dose / concentration required to achieve 50% activation of a biological or biochemical function. The IC50 value or AC50 value of any substance or antagonist can be determined by constructing a dose-response curve and examining the effect of different concentrations of the substance or antagonist on expression levels or functional activity in any assay. IC50 values can be calculated for a given antagonist or substance by determining the concentration needed to inhibit half of the maximum biological response or native expression levels. Thus, the IC50 value for any RNAi construct can be calculated by determining the concentration of the RNAi construct needed to inhibit half of the native INHBE expression level in liver cells (e.g., INHBE expression level in control liver cells) in any assay, such as an immunoassay, RNA FISH assay, or a droplet digital PCR assay. Similarly, AC50 values can be calculated for a given substance by determining the concentration needed to activate half of the maximum biological response or native expression levels. The RNAi constructs disclosed herein may inhibit INHBE expression in liver cells with an IC50 of less than about 20 nM (e.g., less than about 15 nM, 10 nM, 5 nM, or 1 nM). For example, the disclosed RNAi constructs may inhibit INHBE expression in liver cells with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits INHBE expression in liver cells with an ICso of about 1 nM to about 10 nM (e.g., about 5 nM).

[0127] The present disclosure provides methods for reducing or inhibiting expression of INHBE in a subject in need thereof as well as methods of treating or preventing conditions, diseases, or disorders associated with INHBE expression or activity. A “condition, disease, or disorder associated with INHBE expression” refers to conditions, diseases, or disorders in which INHBE expression levels are altered or where elevated expression levels of INHBE are associated with an increased risk of developing the condition, disease, or disorder. As discussed herein, loss of function variants in the INHBE gene are associated with reduced visceral or abdominal adiposity. Abnormally high deposition of visceral adipose tissue is known as “visceral obesity” or “abdominal obesity,” and is associated with disorders such as metabolic syndrome and cardiovascular disease. As such, in certain embodiments, the disclosed RNAi constructs are particularly useful for reducing visceral adiposity, treating or preventing visceral obesity, and treating or preventing cardiovascular disease (e.g. coronary artery disease and myocardial infarction).

[0128] Thus, conditions, diseases, and disorders associated with INHBE expression that can be treated or prevented according to the methods of the disclosure include, but are not limited to, visceral obesity, cardiovascular disease, metabolic syndrome, impaired glucose and lipid metabolism, insulin resistance, and certain cancers (e.g., colon, breast, and prostate) (see, e.g., Shuster et al., Br J Radiol., 85(1009): 1-10 (2012); Ritchie SA and Connell JM., Nutr Metab Cardiovasc Dis; 17:319-26 (2007); Fox et al., Circulation;! 16: 39-48 (2007); Oh et al., J Gastroenterol Hepatol; 23: 411 -17 (2008); and Schapira et al., Cancer; 74: 632-9 (1994)).

[0129] In another aspect, the present disclosure provides uses of a therapeutically effective amount of an RNAi construct disclosed herein for treating a subject, e.g., a subject that would benefit from a reduction and / or inhibition of INHBE gene expression. In a further aspect, the present disclosure provides uses of an RNAi construct, e.g., a siRNA, targeting an INHBE gene or pharmaceutical composition comprising an RNAi construct targeting an INHBE gene in the manufacture of a medicament for treating a subject, e.g., a subject that would benefit from a reduction and / or inhibition of INHBE gene expression and / or INHBE protein production, such as a subject having a disorder that would benefit from reduction in INHBE gene expression, e.g., a INHBE-associated disease.

[0130] The treatment methods (and uses) disclosed herein include administering to the subject, e g., a human, a therapeutically effective amount of any of RNAi constructs described herein, a pharmaceutical composition comprising the RNAi construct, or a vector comprising the RNAi construct. In certain embodiments, the present disclosure provides a method for reducing the expression of INHBE in a patient in need thereof comprising administering to the patient any of the RNAi constructs described herein. The term “patient,” as used herein, refers to a mammal, including humans, and can be used interchangeably with the term “subject.” Preferably, the expression level of INHBE in hepatocytes in the patient is reduced following administration of the RNAi construct as compared to the INHBE expression level in a patient not receiving the RNAi construct or as compared to the INHBE expression level in the patient prior to administration of the RNAi construct. In some embodiments, following administration of an RNAi construct of the disclosure, expression of INHBE is reduced in the patient by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The percent reduction of INHBE expression can be measured by any of the methods described herein as well as others known in the art.

[0131] In some embodiments, a patient in need of reduction of INHBE expression is a patient who has, or its at risk of developing, excess visceral adipose tissue. An “excess” of visceral adipose tissue is an amount greater than a normal level. In clinical practice, excess body fat and obesity are commonly assessed by expressing body weight as a function of height, the most frequently used index being the body mass index (BMI), which is calculated as weight in kilograms divided by height in meter squared. According to the World Health Organization, a BMI over 25 is considered overweight, and a BMI over 30 is generally considered obese. Thus, in some embodiments, the subject has a BMI of at least 30.0 kg / m2(e.g., 30 kg / m2, 30.5 kg / m2, 31 kg / m2, 31.5 kg / m2, 32 kg / m2, 32.5 kg / m2, 33 kg / m2, 33.5 kg / m2, 34 kg / m2, 34.5 kg / m2, 35 kg / m2, 35.5 kg / m2, 36 kg / m2, 36.5 kg / m2, 37 kg / m2, 37.5 kg / m2, 38 kg / m2, 38.5 kg / m2, 39 kg / m2, 39.5 kg / m2, 40 kg / m2). In some embodiments, the subject has a body mass index (BMI) in the range of 30.0 kg / m2to 40.0 kg / m2, inclusive of the endpoints. BMI, however, does not account for the heterogeneity of regional body fat deposition, which has been shown to be a stronger predictor of health risk than overall excessive adiposity (Farkas GJ, Gater DR., J Spinal Cord Med., 41 : 378-87(2018)). The degree or amount ofvisceral adipose tissue may be assessed more directly with techniques including anthropometry, bioelectrical impedance analysis (BIA), dual-energy X-ray absorptiometry (DXA), computed tomography (CT), and magnetic resonance imaging (MRI). In addition to BMI, exemplary anthropometric measurements for assessing visceral fat include waist circumference, hip circumference, and waist-to-hip ratio. In this regard, an excess amount of visceral fat or visceral obesity may be indicated by a waist circumference of 35 inches or greater for females and 40 inches or greater for males, and / or a waist-to-hip ratio greater than 0.85 for females and greater than 0.90 for males. In some embodiments, the subject or patient has a visceral fat percentage of greater than about 10% (e.g., 15%, 20%, 25%, 30%, or greater).

[0132] In certain embodiments, a patient in need of reduction of INHBE expression is a patient who is diagnosed with or at risk of cardiovascular disease. Thus, the present disclosure includes a method for treating or preventing cardiovascular disease in a patient in need thereof by administering any of the RNAi constructs disclosed herein. In some embodiments, the present disclosure includes use of any of the RNAi constructs described herein in the preparation of a medicament for treating or preventing cardiovascular disease in a patient in need thereof. In other embodiments, the present disclosure provides an V / BA’-targeting RNAi construct for use in a method for treating or preventing cardiovascular disease in a patient in need thereof. Cardiovascular disease includes, but is not limited to, myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g. peripheral artery disease), cerebrovascular disease, vulnerable plaque, and aortic valve stenosis. In some embodiments, the cardiovascular disease to be treated or prevented according to the methods of the disclosure is coronary artery disease. In other embodiments, the cardiovascular disease to be treated or prevented according to the methods of the disclosure is myocardial infarction. In yet other embodiments, the cardiovascular disease to be treated or prevented according to the methods of the disclosure is stroke. In still other embodiments, the cardiovascular disease to be treated or prevented according to the methods of the disclosure is peripheral artery disease. In certain embodiments, administration of the RNAi constructs described herein reduces the risk of non-fatal myocardial infarctions, fatal and non- fatal strokes, certain types of heart surgery (e.g., angioplasty, bypass), hospitalization for heart failure, chest pain in patients with heart disease, and / or cardiovascular events in patients with established heart disease (e.g. prior myocardial infarction, prior heart surgery, and / or chest pain withevidence of blocked arteries). In some embodiments, administration of the RNAi constructs described herein according to the methods of the disclosure can be used to reduce the risk of recurrent cardiovascular events.

[0133] In some embodiments, a patient in need of reduction of INHBE expression is a patient who is diagnosed with or at risk of metabolic syndrome. The term “metabolic syndrome,” as used herein, refers to a cluster of conditions that collectively increase the risk of cardiovascular disease, diabetes, and stroke. The cluster of conditions includes, for example, abdominal obesity, insulin resistance, dyslipidemia, and hypertension (Huang, P.L., Dis. Model Meeh.2(5-6): 231-237 (2009)). Thus, the present disclosure includes a method for treating or preventing metabolic syndrome in a patient in need thereof by administering any of the RNAi constructs disclosed herein. In some embodiments, the present disclosure includes use of any of the RNAi constructs described herein in the preparation of a medicament for treating or preventing metabolic syndrome in a patient in need thereof.

[0134] In other embodiments, a patient in need of reduction of INHBE expression is a patient who is diagnosed with or at risk of developing certain types of cancer. Visceral or abdominal obesity has been associated with an increased predisposition to colon, breast, and prostate cancers (see, e.g., Oh et al., J Gastroenterol Hepatol; 23: 411-17 (1994); Schapira et al., Cancer; 74:632-9 (1994), and Von Hafe et al., Obes Res; 12: 1930-5 (2004)). Thus, the present disclosure includes a method for treating or preventing cancer (e.g., colon, breast, or prostate cancer) in a patient in need thereof by administering any of the RNAi constructs disclosed herein. In some embodiments, the present disclosure includes use of any of the RNAi constructs described herein in the preparation of a medicament for treating or preventing cancer in a patient in need thereof.

[0135] Administration of any of the RNAi constructs according to the methods and uses disclosed herein may result in a reduction of the severity, signs, symptoms, and / or markers of such diseases or disorders in a patient with a INHBE-associated disease, e.g., cardiovascular disease. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. Efficacy of treatment or prevention of disease can be assessed, for example, by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect,level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In other embodiments, the efficacy of the methods disclosed herein can be monitored by detecting or monitoring a reduction in visceral adipose tissue and / or a symptom of a INHBE- associated disease. A reduction in visceral adipose tissue may be measured using any anthropomorphic assessment, imaging technique, or other method described herein or known in the art. Exemplary cardiovascular symptoms that may be monitored for reduction or alleviation include, but are not limited to, chest pain, high blood pressure, fatigue, shortness of breath, arrythmia, and dizziness.

[0136] The disclosure further provides methods and uses of any of the disclosed RNAi constructs, or a pharmaceutical composition comprising same, for treating a subject that would benefit from reduction and / or inhibition of INHBE gene expression, e.g., a subject having a INHBE- associated disease, in combination with other therapeutic agents and / or other therapeutic methods, e.g., with known pharmaceuticals and / or known therapeutic methods, such as, for example, those which are currently employed for treating these diseases. For example, in certain embodiments, administration of an RNAi construct targeting an INHBE gene is administered in combination one or more additional pharmaceutical agents (e.g., statins, blood thinners, nitrates, etc.), along with lifestyle changes (e.g., exercise, diet modification, smoking cessation), and / or surgery. The RNAi construct and additional therapeutic agent and / or treatment may be administered at the same time and / or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and / or by another method known in the art or described herein.

[0137] The following examples further illustrate the invention but should not be construed as in any way limiting its scope.EXAMPLE 1

[0138] This example describes the design and synthesis of INHBE RNAi constructs.

[0139] Candidate sequences for the design of therapeutic siRNA molecules targeting the human INHBE gene were identified using a bioinformatics analysis of the human INHBE transcript, thesequence of which is provided herein as SEQ ID NO: 1769 (NCBI Reference Sequence No. NM_031479.5).

[0140] Sequences were also evaluated for cross-reactivity with the INHBE gene from cynomolgus monkeys (NCBI Reference Sequence No. was XM_005571319.2), sequence identity to other human gene sequences and seed region matches to human microRNA (miRNA) sequences to predict off-target effects, and for overlap with known single nucleotide polymorphisms.

[0141] RNAi constructs were synthesized using solid phase phosphoramidite chemistry. Synthesis was performed on MerMadel92X (Bioautomation) instrument. Various chemical modifications, including 2’ -fluoro modified nucleotides, 2’-O-methyl modified nucleotides, abasic nucleotides, and phosphorothioate internucleotide linkages, were incorporated into the molecules. The RNAi constructs were generally formatted to be duplexes of 19-21 base pairs when annealed with either no overhangs (double bluntmer) or one or two overhangs of 2 nucleotides at the 3’ end of the antisense strand and / or the sense strand.Synthesis

[0142] Reagent solutions, phosphoramidite solutions, and solvents were attached to the MerMadel92X instrument. Columns containing solid support (BioAutomation, Universal Support, lumol, 1000 A CPG Solid support) was affixed to the instrument. The columns were washed twice with acetonitrile. The phosphoramidite and reagent solution lines were purged. The synthesis was initiated using the Poseidon software. The synthesis was accomplished by repetition of the deprotection / coupling / oxidation / capping synthesis cycle. Specifically, to the solid support was added detritylation reagent to remove the 5 '-dimethoxy trityl (DMT) protecting group. The solid support was washed with acetonitrile. To the support was added phosphoramidite and activator solution followed by incubation to couple the incoming nucleotide to the free 5 ’-hydroxyl group. The support was washed with acetonitrile. To the support was added oxidation or thiolation reagent to convert the phosphite triester to the phosphate triester or phosphorothioate. To the support was added capping reagents A and B to terminate any unreacted oligonucleotide chains. The support was washed with acetonitrile. After the final reaction cycle, the resin was washed with diethylamine solution to remove the 2-cyanoethyl protecting groups. The support was washed with acetonitrile and dried under vacuum.Cleavage

[0143] The synthesis columns were removed from the synthesizer and affixed to a vacuum manifold with a 96 deep well polypropylene receiving plate. To the solid support was added 250pl of ethanoic ammonium hydroxide 3 : 1 NH4OH: EtOH collecting filtrate in 96 deep well polypropylene plate, repeated three times. The plate was sealed and secured in cleavage block. The cleavage plate was incubated overnight at 55 °C. The block was cooled to less than or equal to room temperature. The plate was removed from block and solution concentrated on a Genevac centrifugal evaporator.Purification and Analysis

[0144] The crude single strand oligonucleotides fractions were desalted by size exclusion chromatography and analyzed by ion pair-reversed phase high-performance liquid chromatographmass spectrometry (HPLC-MS). The desalted single strand oligonucleotides were lyophilized using a Genevac centrifugal evaporator.Annealing

[0145] Using a MinilON (Oxford Nanopore Technologies) automated liquid handler platform, 100p.l of lx phosphate buffered saline (PBS) was added to each well. The plate was sealed and shaken for 5min. Using the liquid handler, a 10: 1 dilution in IxPBS of each single strand oligonucleotide was added to a 96 well Nanoquant plate. The concentration of the single strands were measured using “A260 ssDNA” method.

[0146] Using a TECAN automated liquid handler, the single strand concentrations were imported to dilute single strands to 1 mM and equal volumes of complimentary single strands were combined in a barcoded Matrix tube. The matrix tubes were incubated at 90 °C for 5 minutes and duplexes were analyzed by analytical AEX. The duplex was registered and submitted for in vitro screening.

[0147] The unmodified sense and antisense sequences that were generated are shown in Table 1. Table 2 provides the sequences of the chemically-modified sense and antisense strands. The nucleotide sequences are listed according to the following notations: a, u, g, and c = corresponding 2'-O-methyl ribonucleotide; Af, Uf, Gf, and Cf = corresponding 2 ’-deoxy-2’ -fluoro (“2’-fluoro”)ribonucleotide; and invAb = inverted abasic nucleotide (i.e. abasic nucleotide linked to adjacent nucleotide via a substituent at its 3’ position (a 3’-3’ linkage) when on the 3’ end of a strand or linked to adjacent nucleotide via a substituent at its 5’ position (a 5’-5’ internucleotide linkage) when on the 5’ end of a strand. Insertion of an “s” in the sequence indicates that the two adjacent nucleotides are connected by a phosphorothiodiester group (e.g. a phosphorothioate intemucleotide linkage).Table 1 - Unmodified INHBE siRNA sequencesTable 2 - Modified INHBE siRNA sequencesEXAMPLE 2

[0148] This example demonstrates the efficacy of select INHBE siRNA molecules in RNA FISH assay.

[0149] RNA FISH (fluorescence in situ hybridization) assay was carried out to measure human INHBE mRNA knockdown by siRNAs synthesized in Example 1. Hep3B (ATCC, # HB-8064) cells were cultured in EMEM (ATCC, # 30-2003) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (P-S, Coming). siRNAs were transfected into cells by reverse transfection using Lipofectamine RNAiMAX transfection reagent (Thermo FisherScientific). 1 pL of test siRNAs (in 10 data points doses with 1 :3 dilution starting at 500 nM final concentration) or phosphate-buffered saline (PBS) vehicle and 4 pL of plain EMEM without supplements were added to PDL-coated CellCarrier-384 Ultra assay plates (PerkinElmer) by a Bravo automated liquid handling platform (Agilent). 5 pE of Lipofectamine RNAiMAX (Thermo Fisher Scientific), pre-diluted in plain DMEM without supplements (0.035 pL of RNAiMAX in 5 pL EMEM), was then dispensed into the assay plates by a Multidrop Combi reagent dispenser (Thermo Fisher Scientific). After 20-minute incubation of the siRNA / RNAiMAX mixture at room temperature (RT), 30 pL of Hep3B cells (2000 cells per well) in EMEM supplemented with 10% FBS and 1% P-S were added to the transfection complex using a Multidrop Combi reagent dispenser. The assay plates were incubated at RT for 20 minutes prior to being placed in an incubator. Cells were incubated for 72 hours at 37 °C and 5% CO2.

[0150] RNA FISH assay was performed 72 hours after siRNA transfection using the manufacturer’s assay reagents and protocol (QuantiGene® ViewRNA HC Screening Assay from Thermo Fisher Scientific) on an in-house assembled automated FISH assay platform. In brief, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 minutes at RT, permeabilized with detergent for 3 minutes at RT and then treated with protease solution for 10 minutes at RT. Human INHBE Target-specific probes (Thermo Fisher Scientific, Cat.# VX-02, Assay ID: VA6-3181105- VC) or vehicle (target probe diluent without target probes as negative control) were incubated for 3 hours, whereas preamplifiers, amplifiers, and label probes were incubated for 1 hour each. All hybridization steps were carried out at 40 °C in a Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific).

[0151] After hybridization reactions, cells were stained for 30 minutes with Hoechst and CellMask Blue (Thermo Fisher Scientific) and then imaged on an Opera Phenix high-content screening system (PerkinElmer). The images were analyzed using a Columbus image data storage and analysis system (PerkinElmer) to obtain the mean spot count per cell. The mean spot count per cell was normalized using the high (PBS with target probes) and low (PBS without target probes) control wells. The high and low controls have normalized values of 100 and 0, respectively. The normalized values against the test siRNA concentrations were fitted to a 4-parameter sigmoidal model using Genedata Screener data analysis software (Genedata, Basel, Switzerland) to obtain IC50 values and maximum activity.

[0152] The results of the assay are shown in Table 3. INHBE-targeted siRNA activity is expressed as a percentage of INHBE mRNA knockdown compared to control. Negative values indicate a decrease in INHBE mRNA levels.Table 3. In vitro inhibition of IHHBE mRNA in Human Hepatocytes

[0153] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0154] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. 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. Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0155] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS:

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides of a sequence selected from the antisense sequences listed in Table 1 and Table 2, wherein the RNAi construct inhibits the expression of an inhibin subunit beta E (INHBE) mRNA sequence.

2. The RNAi construct of claim 1, wherein the sense strand comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length.

3. The RNAi construct of claim 2, wherein the duplex region is about 17 to about 24 base pairs in length.

4. The RNAi construct of claim 2 or claim 3, wherein the duplex region is about 19 to about 21 base pairs in length.

5. The RNAi construct of any one of claims 1-4, wherein the sense strand and the antisense strand are each independently about 19 to about 27 nucleotides in length.

6. The RNAi construct of claim 5, wherein the sense strand and the antisense strand are each independently about 19 to about 23 nucleotides in length.

7. The RNAi construct of any one of claims 1-6, which comprises one or two blunt ends.

8. The RNAi construct of any one of claims 1-7, which comprises one or two nucleotide overhangs of 1 to 4 unpaired nucleotides.

9. The RNAi construct of claim 8, wherein the nucleotide overhang comprises two unpaired nucleotides.

10. The RNAi construct of claim 8 or 9, wherein the RNAi construct comprises a nucleotide overhang at the 3’ end of the sense strand, the 3’ end of the antisense strand, or the 3’ end of both the sense strand and the antisense strand.

11. The RNAi construct of any one of claims 80, wherein the nucleotide overhang comprises a 5’-UU-3’ dinucleotide or a 5’-dTdT-3’ dinucleotide.

12. The RNAi construct of any one of claims 11, wherein the RNAi construct comprises at least one modified nucleotide.

13. The RNAi construct of claim 12, wherein the modified nucleotide is a 2’-modified nucleotide.

14. The RNAi construct of claim 12, wherein the modified nucleotide is a 2’-fluoro modified nucleotide, a 2’-O-methyl modified nucleotide, a 2’ -O-m ethoxy ethyl modified nucleotide, a 2’-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a deoxyribonucleotide, or combinations thereof.

15. The RNAi construct of claim 13, wherein the modified nucleotide is a 2’-O-methyl modified nucleotide, a 2’ -O-m ethoxy ethyl modified nucleotide, a 2’-fluoro modified nucleotide, or combinations thereof.

16. The RNAi construct of any one of claims 125, wherein all of the nucleotides in the sense and antisense strands are modified nucleotides.

17. The RNAi construct of claim 16, wherein the modified nucleotides are 2’-O-methyl modified nucleotides, 2’-fluoro modified nucleotides, or combinations thereof.

18. The RNAi construct of any one of claims 17, wherein the sense strand comprises an abasic nucleotide as the terminal nucleotide at its 3’ end, its 5’ end, or both its 3’ and 5’ ends.

19. The RNAi construct of claim 18, wherein the abasic nucleotide is linked to an adjacent nucleotide through a 3 ’-3’ internucleotide linkage or a 5 ’-5’ internucleotide linkage.

20. The RNAi construct of any one of claims 19, wherein the sense strand, the antisense strand, or both the sense and antisense strands comprise one or more phosphorothioate internucleotide linkages.

21. The RNAi construct of claim 20, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3’ end of the antisense strand.

22. The RNAi construct of claim 20, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3’ and 5’ ends of the antisense strand.

23. The RNAi construct of claim 20, wherein RNAi construct comprises at least one phosphorothioate intemucleotide linkage at the 3’ end and / or the 5’ end of the sense strand.

24. The RNAi construct of claim 23, wherein the sense strand comprises two consecutive phosphorothioate intemucleotide linkages between the terminal nucleotides at the 5’ end.

25. The RNAi construct of any one of claims 1-24, wherein the antisense strand comprises or consists of a sequence selected from the antisense sequences listed in Table 1 and Table 2.

26. The RNAi construct of any one of claims 1-25, wherein the antisense strand comprises or consists of a sequence selected from SEQ NO: 642, SEQ ID NO: 660, SEQ ID NO: 681, SEQ ID NO: 701, SEQ ID NO: 1526, SEQ ID NO: 1544, SEQ ID NO: 1565, and SEQ ID NO: 1585.

27. The RNAi construct of any one of claims 1-26, wherein the sense strand comprises or consists of a sequence selected from the sense sequences listed in Table 1 or Table 2.

28. The RNAi construct of claim 27, wherein the sense strand comprises or consists of a sequence selected from SEQ ID NO: 200, SEQ ID NO: 218, SEQ ID NO: 239, SEQ ID NO: 259, SEQ ID NO: 1084, SEQ ID NO: 1102, SEQ ID NO: 1123, and SEQ ID NO: 1143.

29. The RNAi construct of any one of claims 1-28, wherein the RNAi construct is any one of the duplex compounds listed in Table 1 or Table 2.

30. The RNAi construct of claim 29, wherein:(a) the sense strand comprises or consists of the sequence of SEQ ID NO: 200 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 642;(b) the sense strand comprises or consists of the sequence of SEQ ID NO: 1084 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1526;(c) the sense strand comprises or consists of the sequence of SEQ ID NO: 218 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 660;(d) the sense strand comprises or consists of the sequence of SEQ ID NO: 1102 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1544;(e) the sense strand comprises or consists of the sequence of SEQ ID NO: 239 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 681;(f) the sense strand comprises or consists of the sequence of SEQ ID NO: 1123 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1565;(g) the sense strand comprises or consists of the sequence of SEQ ID NO: 259 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 701; or(h) the sense strand comprises or consists of the sequence of SEQ ID NO: 1143 and the antisense strand comprises or consists of the sequence of SEQ ID NO: 1585.

31. The RNAi construct of any one of claims 1-30, further comprising a ligand.

32. The RNA construct of claim 31, wherein the ligand binds to one or more proteins expressed on the surface of hepatocytes.

33. The RNAi construct of claim 32, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folate moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or antigen-binding fragment thereof.

34. The RNAi construct of claim 33, wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine (GalNAc).

35. A composition comprising the RNAi construct of any one of claims 1-34 and a pharmaceutically acceptable carrier, excipient, or diluent.

36. A method for reducing the expression of INHBE in a patient in need thereof comprising administering to the patient the RNAi construct of any one of claims 1-34 or the composition of claim 36.

37. A method for reducing visceral adiposity in a patient in need thereof comprising administering to the patient the RNAi construct of any one of claims 1-34 or the composition of claim 36.

38. The method of claim 36 or 37, wherein the patient is diagnosed with or at risk for cardiovascular disease.

39. A method for treating or preventing cardiovascular disease in a patient in need thereof comprising administering to the patient the RNAi construct of any one of claims 1-34 or the composition of claim 36.

40. The method of claim 38 or 39, wherein the cardiovascular disease is coronary artery disease, peripheral artery disease, myocardial infarction, or stroke.

41. The method of any one of claims 36-40, wherein the expression level of INHBE in hepatocytes is reduced in the patient following administration of the RNAi construct as compared to the INHBE expression level in a patient not receiving the RNAi construct.

42. The method of any one of claims 36-41, wherein the patient has a visceral fat percentage of 10% or greater.

43. An RNAi construct of any one of claims 1-34 or the composition of claim 36 for reducing the expression of INHBE in a patient in need thereof.

44. An RNAi construct of any one of claims 1-34 or the composition of claim 36 for use in a method of treating cardiovascular disease in a patient in need thereof.

45. The RNAi construct of claim 44, wherein the cardiovascular disease is coronary artery disease, peripheral artery disease, myocardial infarction, or stroke.

46. An RNAi construct of any one of claims 1-34 or the composition of claim 36 for use in a method of reducing visceral adiposity in a patient in need thereof.

47. The RNAi construct of any one of claims 38-46, wherein the patient has a visceral fat percentage of 10% or greater.

48. Use of an RNAi construct of any one of claims 1-34 or the composition of claim 35 for the preparation of a medicament for treating cardiovascular disease in a patient in need thereof.

49. The use of claim 48, wherein the cardiovascular disease is coronary artery disease, peripheral artery disease, myocardial infarction, or stroke.

50. Use of an RNAi construct of any one of claims 1-34 or the composition of claim 36 for reducing expression of INHBE in a patient in need thereof.

51. Use of an RNAi construct of any one of claims 1-34 or the composition of claim 36 for reducing visceral adiposity in a patient in need thereof.

52. The use of any one of claims 48-51, wherein the patient has a visceral fat percentage of 10% or greater.