Compositions and methods for modulating SCAP activity

Abstract

Oligonucleotides and compositions comprising same that modulate (e.g., inhibit, limit, or reduce) sterol regulatory element binding protein (SREBP) cleavage and activating protein (SCAP) activity. Methods of making and using the oligonucleotides are also disclosed, particularly in connection with treating diseases, disorders, and / or conditions associated with SCAP activity, such as nonalcoholic fatty liver disease (NAFLD), (NASH), dyslipidemia, atherosclerotic cardiovascular disease (ASCVD), and / or other SCAP-associated conditions, diseases, and / or disorders.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 363,091, filed April 15, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to biology and medicine, and more specifically to oligonucleotides and compositions comprising same for modulating (e.g., inhibiting or reducing) sterol regulatory element binding protein (SREBP) cleavage activating protein (SCAP) activity, and their use for treating conditions, diseases, and / or disorders associated with SCAP. [Background technology]

[0003] SCAP is a cholesterol-binding endoplasmic reticulum (ER) membrane protein that binds to SREBP transcription factors and transports them from the ER to the Golgi apparatus for processing. Once processed in the Golgi apparatus, the SREBP transcription factors translocate to the nucleus, where they are involved in the regulation of genes involved in lipid homeostasis. SREBP1a, SREBP1c, and SREBP2 are regulated by SREBP cleavage-activating protein (SCAP), which is encoded by the SCAP gene. SREBP1c is the most abundant SREBP in the liver, and its regulation is important for maintaining lipid homeostasis. Specifically, SREBPs affect lipid homeostasis by regulating genes involved in lipid biosynthesis, as well as genes involved in lipid clearance, such as low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin / kexin type (PCSK9). SCAP is also involved in NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation.

[0004] Human SCAP is expressed ubiquitously throughout the body, with the highest protein expression in bone marrow, brain, endocrine tissues, gastrointestinal tract, liver, lymphoid tissues, muscle tissues, pancreas, reproductive organs, and the airways. The role that SCAP plays in regulating the transcription of genes involved in lipid homeostasis makes SCAP a promising therapeutic target to attenuate the progression of NASH at various stages.

[0005] Although several therapeutic agents against SCAP exist, there is a need for additional therapeutic agents to inhibit or reduce SCAP activity for treating liver diseases, particularly NAFLD and nonalcoholic steatohepatitis (NASH). Summary of the Invention

[0006] To address this need, the present disclosure describes compositions and methods for treating diseases, disorders, and / or conditions associated with SCAP activity. The present disclosure is based, in part, on the discovery and development of double-stranded (ds) oligonucleotides (e.g., RNAi oligonucleotides) for selectively modulating (e.g., inhibiting and / or reducing) SCAP activity, for example, in the liver. Thus, target sequences within SCAP are identified, and RNAi oligonucleotides are generated that bind to these target sequences and inhibit SCAP mRNA expression. As shown herein, some oligonucleotides inhibit SCAP activity in at least humans and non-human primates (NHPs) (i.e., double common), and other oligonucleotides inhibit SCAP activity in mice, humans, and NHPs in the liver (i.e., triple common). Without being bound by theory, the RNAi oligonucleotides herein are useful for treating diseases, disorders, and / or conditions associated with SCAP activity (e.g., liver diseases such as NAFLD, NASH, dyslipidemia, and / or atherosclerotic cardiovascular disease (ASCVD)).

[0007] Thus, the present disclosure describes an RNAi oligonucleotide for reducing or inhibiting SCAP activity comprising a sense strand (also known as a passenger strand) and / or an antisense strand (also known as a guide strand), wherein the sense strand has a structure set forth in Table 3, and the antisense strand has a sequence set forth in Table 3.

[0008] In some embodiments, the sense strand has a sequence set forth in Table 3 (eg, any one of the odd numbers of SEQ ID NOs: 9-392), particularly any one of SEQ ID NOs: 139, 147, 221, 273, 321, 333, and 361.

[0009] In some embodiments, the antisense strand has a sequence set forth in Table 3 (eg, any one of the even numbers of SEQ ID NOs: 9-392), particularly any one of SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362.

[0010] Alternatively, the present disclosure describes an RNAi oligonucleotide for reducing or inhibiting SCAP activity comprising a sense strand and / or an antisense strand, wherein the sense strand has a sequence set forth in Table 4 and the antisense strand has a sequence set forth in Table 4.

[0011] In some embodiments, the sense strand has a sequence set forth in Table 4 (eg, any one of the odd numbers of SEQ ID NOs: 393-776), particularly any one of SEQ ID NOs: 523, 531, 605, 657, 705, 717, and 745.

[0012] In some embodiments, the antisense strand has a sequence set forth in Table 4 (eg, any one of the even numbers of SEQ ID NOs: 393 to 776), particularly any one of SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746.

[0013] Alternatively, an RNAi oligonucleotide for reducing or inhibiting SCAP activity is described, which comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, and the antisense strand has a region of complementarity to any one of SCAP mRNA target sequences of SEQ ID NOs: 777 to 783.

[0014] In any of the above embodiments, the sense strand is about 15 nucleotides to about 50 nucleotides in length (or nucleotides in length). In some embodiments, the sense strand is about 20 nucleotides to about 40 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length.

[0015] In any of the above embodiments, the antisense strand is about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the antisense strand is about 20 nucleotides to about 25 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length.

[0016] In any of the above embodiments, the duplex region is about 19 nucleotides to about 21 nucleotides in length. In some embodiments, the duplex region is 20 nucleotides in length.

[0017] In any of the above embodiments, the region of complementarity is at least 15 contiguous nucleotides in length. In some embodiments, the region of complementarity is at least 19 contiguous nucleotides in length to at least 21 contiguous nucleotides in length. In other embodiments, the region of complementarity is 19 contiguous nucleotides in length or 21 contiguous nucleotides in length.

[0018] In any of the above embodiments, the RNAi oligonucleotide comprises a 3'-terminal stem loop on the sense strand shown as S1-L-S2, where a first stem portion (S1) is complementary to a second stem portion (S2), and L is a loop between S1 and S2 about 3 to about 5 nucleotides in length.

[0019] In any of the above embodiments, the antisense strand, the sense strand, or both have an overhang sequence.In some embodiments, the antisense strand comprises a 3' overhang that is one or more nucleotides in length.In other embodiments, the 3' overhang sequence is 2 nucleotides in length, for example, GG.

[0020] The oligonucleotides are also described as comprising an antisense strand and a sense strand for reducing or inhibiting SCAP activity, wherein the antisense strand can be from about 21 nucleotides to about 27 nucleotides in length and has a region of complementarity to SCAP mRNA, and the sense strand comprises a stem-loop at its 3' end shown as S1-L-S2, where S1 is complementary to S2 and L forms a loop between S1 and S2 about 3 to about 5 nucleotides in length, and the antisense strand and the sense strand form a duplex structure at least about 19 nucleotides in length but are not covalently linked.

[0021] In some embodiments, loop L is a triloop (triL) or a tetraloop (tetraL). In some embodiments, L is a tetraL that is four nucleotides in length. In particular embodiments, L is a tetraL having the sequence 5'-GAAA-3'.

[0022] In some embodiments, S1 and S2 are 1-10 nucleotides in length and have the same length. In other embodiments, S1 and S2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides in length. In other embodiments, S1 and S2 are 6 nucleotides in length. In certain embodiments, the stem loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 784).

[0023] In some embodiments, the sense strand is 25 nucleotides in length and the antisense strand is 27 nucleotides in length, in other embodiments, the sense strand is 36 nucleotides in length and the antisense strand is 22 nucleotides in length.

[0024] In the above embodiments, the duplex region comprises a 3' overhanging sequence on the antisense strand. In some embodiments, the 3' overhanging sequence on the antisense strand is 2 nucleotides in length.

[0025] In any of the above embodiments, at least one nucleotide in the oligonucleotide is a modified nucleotide.In some embodiments, all nucleotides in the oligonucleotide are modified except for the nucleotide in the stem loop (i.e., S1-L-S2).In other embodiments, all nucleotides in the oligonucleotide are modified except for the nucleotide in the loop L.

[0026] In some embodiments, modified nucleotides include 2'-modifications such as, for example, 2'-aminoethyl (EA), 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), and 2'-deoxy-2'-fluoro-β-arabinonucleic acid (2'-FANA). In certain embodiments, all nucleotides in the oligonucleotide include a 2'-modification, for example, 2'-F or 2'-OMe. In some embodiments, about 18% to about 23%, or 18%, 19%, 20%, 21%, 22%, or 23% of the nucleotides in the sense strand include a 2'-F modification. In other embodiments, about 38% to about 43%, or 38%, 39%, 40%, 41%, 42%, or 43% of the nucleotides in the sense strand include a 2'-F modification. In some embodiments, about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2'-F modification. In some embodiments, about 25% to about 35%, or 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise a 2'-F modification. In some embodiments, about 35% to about 45%, or 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45% of the nucleotides of the oligonucleotide comprise a 2'-F modification.

[0027] In any of the above embodiments, at least one nucleotide in the oligonucleotide comprises a modified internucleotide linkage. In some embodiments, the modified internucleotide linkage is a phosphorothioate (PS) linkage.

[0028] In any of the above embodiments, the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand includes a phosphate analog, such as, for example, oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. Alternatively, or optionally, the phosphate analog is a 4'-phosphate analog, including 5'-methoxyphosphonate-4'-oxy.

[0029] In any of the above embodiments, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, such as, for example, amino sugars, carbohydrates, cholesterol, lipids, or polypeptides. In some embodiments, the targeting ligand is an N-acetylgalactosamine (GalNAc) moiety. In other embodiments, the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

[0030] In some embodiments, the targeting ligand is conjugated to one or more nucleotides of L of the stem loop. In certain embodiments, up to four nucleotides of the stem loop are each conjugated to a monovalent GalNac moiety.

[0031] In certain embodiments, one or more nucleotides at positions 8, 9, 10, or 11 of the sense strand are modified with 2'-F. In other embodiments, the sugar moiety of each of the nucleotides at positions 1-7, 12-27, and 31-36 of the sense strand is modified with 2'-OMe. In certain embodiments, the nucleotides at positions 8-11 of the sense strand are modified with 2'-F, and positions 1-7, 12-27, and 31-36 are modified with 2'-OMe.

[0032] In other specific embodiments, the antisense strand has one or more nucleotides at positions 2-5, 7, 10, and 14 modified with 2'-F and one or more nucleotides at positions 1, 6, 8-9, 11-13, and 15-22 modified with 2'-OMe. In other embodiments, the antisense strand contains 2'-F modified nucleotides at positions 2-5, 7, 10, and 14 and 2'-OMe modified nucleotides at positions 1, 6, 8-9, 11-13, and 15-22.

[0033] In certain embodiments, the oligonucleotide has a modification pattern as shown in FIG.

[0034] In any of the above embodiments, the oligonucleotide is an RNAi oligonucleotide. In some embodiments, the RNAi oligonucleotide comprises a sense strand having a nucleotide sequence shown in Table 3, particularly any one of SEQ ID NOs: 139, 147, 221, 273, 321, 333, and 361. In certain embodiments, the RNAi oligonucleotide comprises a sense strand having a nucleotide sequence shown in Table 4, particularly any one of SEQ ID NOs: 523, 531, 605, 657, 705, 717, and 745. In some embodiments, the RNAi oligonucleotide comprises an antisense strand having a nucleotide sequence shown in Table 3, particularly any one of SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362. In certain embodiments, the RNAi oligonucleotide comprises an antisense strand having a nucleotide sequence shown in Table 4, particularly any one of SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746.

[0035] In certain embodiments, the RNAi oligonucleotide comprises a sense strand having a nucleotide sequence of any one of SEQ ID NOs: 139, 147, 221, 273, 321, 333, and 361, and an antisense strand having a nucleotide sequence of any one of SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362.

[0036] In other specific embodiments, the sense and antisense strands of the RNAi oligonucleotide are each selected from the following: (a) SEQ ID NOs: 139 and 140; (b) SEQ ID NOs: 147 and 148; (c) SEQ ID NOs: 221 and 222; (d) SEQ ID NOs: 273 and 274; (e) SEQ ID NOs: 321 and 322; (f) SEQ ID NOs: 333 and 334; (g) SEQ ID NOs: 361 and 362.

[0037] In certain embodiments, the RNAi oligonucleotide comprises a sense strand having the nucleotide sequence of any one of SEQ ID NOs: 147 and 333, and an antisense strand having the nucleotide sequence of any one of SEQ ID NOs: 148 and 334, respectively.

[0038] In certain embodiments, the RNAi oligonucleotides comprise a sense strand having a nucleotide sequence of any one of SEQ ID NOs: 523, 531, 605, 657, 705, 717, and 745, and an antisense strand having a nucleotide sequence of any one of SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746, respectively.

[0039] In other specific embodiments, the sense and antisense strands of the RNAi oligonucleotide are each selected from the following: (a') SEQ ID NOs: 523 and 524; (b') SEQ ID NOs: 531 and 532; (c') SEQ ID NOs: 605 and 606; (d') SEQ ID NOs: 657 and 658; (e') SEQ ID NOs: 705 and 706; (f') SEQ ID NOs: 717 and 718; (g') SEQ ID NOs: 745 and 746;

[0040] In certain embodiments, the RNAi oligonucleotide comprises a sense strand having the nucleotide sequence of any one of SEQ ID NOs: 531 and 717, and an antisense strand having the nucleotide sequence of any one of SEQ ID NOs: 532 and 718, respectively.

[0041] Also described is an RNAi oligonucleotide for inhibiting or reducing SCAP activity, comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex region, wherein all nucleotides of the sense strand and the antisense strand comprise base, sugar, and / or internucleotide bond modifications, and wherein the antisense strand comprises a region of complementarity to any one of SCAP mRNA target sequences of SEQ ID NOs: 777-783, wherein the region of complementarity is at least about 15 contiguous nucleotides in length.

[0042] In another aspect, a pharmaceutical composition is described, comprising at least one oligonucleotide herein, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, delivery agent, or excipient. In some embodiments, the pharmaceutical composition includes an additional therapeutic agent, such as, for example, a lipid-lowering agent, an antidiabetic agent, or an antiobesity agent.

[0043] In another aspect, a method for reducing SCAP activity in a cell, a population of cells, a tissue, an organ, or an individual is described, the method comprising at least the step of administering / contacting the oligonucleotide herein or the pharmaceutical composition herein to the cell, the population of cells, the tissue, the organ, or the individual. In some embodiments, reducing SCAP activity includes reducing the amount or level of SCAP mRNA, the amount or level of SCAP protein, SCAP activity, or a combination thereof in the cell, the population of cells, the tissue, the organ, or the individual. In some embodiments, the cell, the population of cells, the tissue, the organ, or the individual has a disease, disorder, or condition associated with SCAP activity. In certain embodiments, the disease, disorder, or condition associated with SCAP activity is NAFLD, NASH, dyslipidemia, and / or ASCVD.

[0044] In another aspect, a method for treating an individual having or suspected of having a disease, disorder, or condition associated with SCAP activity is described. The method includes at least a step of administering an effective amount of the oligonucleotide herein or the pharmaceutical composition herein to an individual in need thereof. In some embodiments, the disease, disorder, or condition associated with SCAP activity is NAFLD, NASH, dyslipidemia, and / or ASCVD. In some embodiments, the oligonucleotide or pharmaceutical composition is administered by subcutaneous (SQ) administration daily, weekly, monthly, quarterly, yearly, particularly monthly or quarterly.

[0045] In some embodiments, the individual has alcoholic hepatitis (AH), alcoholic liver disease (ALD), cholangiocarcinoma (CCA), cirrhosis, liver fibrosis, liver inflammation, hepatocellular carcinoma (HCC), fatty liver, NAFLD, NASH, primary sclerosing cholangitis (PSC), hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, diabetes, and / or obesity, and / or ASCVD.

[0046] In any of the above embodiments, the method may include additional steps such as measuring or obtaining genotype information, SCAP mRNA, SCAP3 protein levels, SCAP activity, the individual's body weight and / or blood glucose levels and / or cholesterol and / or TG, and then comparing the obtained values ​​to one or more baseline or previously obtained values ​​to assess the effectiveness of the contacting or administering.

[0047] In any of the above embodiments, the method includes administering the RNAi oligonucleotide or pharmaceutical composition simultaneously, separately, or sequentially with the second composition or second therapeutic agent. In some embodiments, the second composition or second therapeutic agent is a SCAP antibody or fragment thereof, a lipid-lowering agent, an antidiabetic agent, or an antiobesity agent. In some embodiments, the second composition or second therapeutic agent is administered at the same frequency as the RNAi oligonucleotide (i.e., every other day, twice a week, or weekly). In other embodiments, the second composition or second therapeutic agent is administered at a different frequency than the RNAi oligonucleotide. Similarly, in other embodiments, the second composition or second therapeutic agent is administered by the same route as the RNAi oligonucleotide (e.g., SQ). In still other embodiments, the second composition or second therapeutic agent is administered by a different route than the RNAi oligonucleotide.

[0048] In another aspect, the use of the RNAi oligonucleotides herein, optionally administered simultaneously, separately or sequentially (i.e., in combination) with a second composition or a second therapeutic agent, for treating a disease, disorder or condition associated with SCAP activity is described.

[0049] In another aspect, the present specification describes the use of the RNAi oligonucleotides in the manufacture of a medicament for treating a disease, disorder or condition associated with SCAP activity, the medicament optionally further comprising a second composition or a second therapeutic agent.

[0050] In another aspect, a kit is described comprising at least one oligonucleotide of the present specification, an optional pharma- ceutically acceptable carrier, and a package insert containing instructions for administering the same to an individual having a disease, disorder, or condition associated with SCAP activity.

[0051] An advantage of the oligonucleotides and compositions herein is that inhibiting SCAP activity exerts beneficial effects on the full range of NAFLD, NASH, dyslipidemia, and / or ASCVD.

[0052] Further advantages, benefits, features, objects and the like will become more apparent from a consideration of the following detailed description, such detailed description referring to the following drawing(s). [Brief description of the drawings]

[0053] [Figure 1] 1 discloses a schematic diagram showing the structure and chemical modification pattern of a typical GalNAc-conjugated SCAP oligonucleotide (modification pattern M1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] overview NAFLD and NASH are significant public health burdens because they are chronic liver disorders that begin with hepatic TG accumulation (steatosis) and progress to hepatic inflammation and fibrosis, cirrhosis, and even hepatocellular carcinoma. SCAP is a transcriptional regulator that has been shown to be associated with NAFLD and NASH. Here, targeted silencing of SCAP mRNA via RNAi can prevent the processing and downstream transcriptional changes of active SREBP in regulating de novo lipogenesis and TG accumulation in the liver.

[0055] RNAi is the process of introducing exogenous RNA into a cell to specifically degrade the mRNA that codes for a targeted protein, resulting in decreased expression of the target gene.

[0056] In humans, SCAP is 1279 amino acids long with a predicted molecular weight of 140 kD. Exemplary nucleic acid sequences for SCAP can be found in the NCBI reference sequences NM_012235 (isoform 1) and NM_001320044 (isoform 2) (human); NM_001001144 and NM_001103162 (mouse); NM_001100966 (rat); and XM_001100342 (primate). Other exemplary nucleic acid sequences of SCAP include those set forth in NCBI Reference SEQ ID NOs: XM_017005918 (human variant X1), XM_011533501 (human variant X2), XM_005264967 (human variant X3), XM_005264968 (human variant X4), XM_011533502 (human variant X5), XM_005264971 (human variant X6), XM_017005921 (human variant X7), XM_006512083 (mouse variant X1), XM_006512084 (mouse variant X2), XM_006512085 (mouse variant X3), XM_006512086 (mouse variant X4), XM_006512087 (mouse variant X5), XM_006512088 (mouse variant X6), XM_006512089 (mouse variant X7), XM_006512090 (mouse variant X8), XM_006512091 (mouse variant X9), XM_006512092 (mouse variant X10), XM_006512093 (mouse variant X11), XM_006512094 (mouse variant X12), XM_006512095 (mouse variant X13), XM_006512096 (mouse variant X14), XM_006512097 (mouse variant X15), XM_006512098 (mouse variant X16), XM_006512099 (mouse variant X17), XM_006512010 (mouse variant X18), XM_00651201 6243922 (rat variant X1), XM_017595596 (rat variant X2), XM_006243923 (rat variant X3), XM_006243924 (rat variant X5), XM_006243925 (rat variant X5), XM_017595597 (rat variant X6), XM_005546961 (primate variant X1), XM_015445807 (primate variant X2), XM_005546962 (primate variant X3), XM_005546963 (primate variant X4), and XM_015445808 (primate variant X5). However, one of skill in the art will appreciate that additional examples of SCAP mRNA sequences are readily available using public databases such as GenBank and UniProt.

[0057] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In carrying out or testing the RNAi oligonucleotides, pharmaceutical compositions containing RNAi oligonucleotides, and methods of making and using such RNAi oligonucleotides herein, any methods and materials similar or equivalent to those described herein can be used, but the preferred methods and materials are described herein.

[0058] Furthermore, reference to an element with the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that only one or only one of the element is present. Thus, the indefinite article "a" or "an" normally means "at least one."

[0059] Furthermore, use of the term "including" as well as other forms such as "include," "includes," and "included" are not limiting.

[0060] Certain definitions as used herein are defined as follows:

[0061] As used herein, "about" means within a statistically meaningful range of a value or values, for example, a specified concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such values ​​or ranges are typically within an order of magnitude of 20%, more typically within 10%, and even more commonly within 5% of a given value or range. The allowable variation encompassed by "about" depends on the particular system under study and is readily apparent to one of ordinary skill in the art.

[0062] As used herein, "administer," "administering," "administration," and the like refer to providing a substance (e.g., an oligonucleotide herein or a composition herein) to an individual in a pharmacologically useful manner (e.g., to treat a disease, disorder, or condition of the individual).

[0063] As used herein, "antisense strand" refers to an oligonucleotide herein that is complementary to a region of a target sequence. Similarly, as used herein, "sense strand" refers to an oligonucleotide herein that is complementary to a region of the antisense strand.

[0064] As used herein, "asialoglycoprotein receptor" or "ASGPR" refers to a bipartite C-type lectin formed by a 48 kDa major subunit (ASGPR-1) and a 40 kDa minor subunit (ASGPR-2). ASGPR plays a primary role in binding, internalization, and subsequent excretion of circulating glycoproteins containing terminal galactose or GalNAc residues (asialoglycoproteins), which are expressed primarily on the sinusoidal surface of hepatocytes.

[0065] As used herein, "attenuate," "attenuate," "attenuate," and the like refer to reducing or effectively stopping. As a non-limiting example, one or more of the treatments herein can reduce or effectively stop the onset or progression of AH, ALD, CCA, cirrhosis, liver fibrosis, hepatitis, HCC, fatty liver, NAFLD, NASH, and PSC, as well as related diseases, disorders, and conditions in an individual, such as, for example, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, ASCVD, diabetes, and / or obesity. This attenuation can include a reduction in one or more aspects (e.g., symptoms, tissue characteristics, and cellular, inflammatory, or immunological activity) of associated diseases, disorders, and conditions in an individual, such as, for example, AH, ALD, CCA, cirrhosis, liver fibrosis, hepatitis, HCC, fatty liver, NAFLD, NASH, and PSC, and for example, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, ASCVD, diabetes, and / or obesity; absence of detectable progression (worsening) of associated diseases, disorders, and conditions in an individual, such as hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, ASCVD, diabetes, and / or obesity; absence of detection of AH, ALD, CCA, cirrhosis, liver fibrosis, hepatitis, HCC, fatty liver, NAFLD, NASH, and PSC, as well as associated diseases, disorders, and conditions in an individual, such as, for example, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, ASCVD, diabetes, and / or obesity, when otherwise expected.

[0066] As used herein, "complementary" refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that allows the two nucleotides to base pair with each other. For example, purine nucleotides of one nucleic acid that are complementary to pyrimidine nucleotides of an opposing nucleic acid can base pair together by forming hydrogen bonds with each other. Complementary polynucleotide strands can base pair in a Watson-Crick fashion or in any other manner that allows for the formation of a stable duplex. Similarly, two nucleic acids can have regions of multiple nucleotides that are complementary to each other, thereby forming a region of complementarity, as described herein.

[0067] As used herein, "contact," "contacting," and the like refer to directly or indirectly introducing or delivering an oligonucleotide, such as an RNAi oligonucleotide, into a cell, for example by promoting or effecting uptake or absorption into the cell.

[0068] As used herein, "deoxyribonucleotide" refers to a nucleotide that has a hydrogen in place of the hydroxyl at the 2' position of its pentose sugar when compared to a ribonucleotide. Modified deoxyribonucleotides have one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions within or of the nucleobase, sugar, or phosphate group.

[0069] As used herein, "double-stranded oligonucleotide" or "ds oligonucleotide" refers to an oligonucleotide that is substantially in double-stranded form. The complementary base pairing of the duplex region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of covalently separated nucleic acid strands. Similarly, the complementary base pairing of the duplex region(s) of a ds oligonucleotide can be formed between antiparallel sequences of nucleotides of covalently linked nucleic acid strands. Furthermore, the complementary base pairing of the duplex region(s) of a ds oligonucleotide can be formed from a single nucleic acid strand that folds (e.g., via a hairpin) and provides a complementary antiparallel sequence of nucleotides that base pair together. A ds oligonucleotide can include two covalently separated nucleic acid strands that are fully double-stranded with each other. However, a ds oligonucleotide can include two covalently separated nucleic acid strands that are partially double-stranded (e.g., with an overhang at one or both ends). A ds oligonucleotide may contain antiparallel sequences of nucleotides that are partially complementary and therefore may have one or more mismatches, which may include internal or terminal mismatches.

[0070] As used herein, "duplex" with respect to nucleic acids (eg, oligonucleotides) means the structure formed by complementary base pairing of two antiparallel sequences of nucleotides.

[0071] As used herein, "excipient" means a non-therapeutic agent that may be included in the compositions herein to, for example, impart or contribute a desired consistency or stabilizing effect.

[0072] As used herein, "hepatocyte" or "plurality of hepatocytes" refers to cells of the liver parenchyma. These cells make up approximately 70%-85% of the liver's mass and produce serum albumin, fibrinogen (FBN), and the prothrombin group of clotting factors (except factors 3 and 4). Markers of hepatocyte lineage cells include, but are not limited to, transthyretin (Ttr), glutamine synthetase (GluI), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers of mature hepatocytes include, but are not limited to, cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, e.g., Huch et al. (2013) Nature 494:247-250.

[0073] As used herein, "hepatotoxic agent" means a compound, virus, or other substance that is itself toxic to the liver or that can be processed to form metabolic products that are toxic to the liver. Hepatotoxic substances include carbon tetrachloride (CCl 4 ), acetaminophen (paracetamol), vinyl chloride, arsenic, chloroform, and nonsteroidal anti-inflammatory drugs (such as aspirin and phenylbutazone).

[0074] As used herein, "individual" refers to any mammal, including cats, dogs, mice, rats, and primates, particularly humans. "Subject" or "patient" may be used interchangeably with "individual."

[0075] As used herein, a "labile linker" refers to a linker that can be cleaved (e.g., by acidic pH). Similarly, a "fairly stable linker" refers to a linker that cannot be cleaved.

[0076] As used herein, "liver inflammation" or "hepatitis" refers to a physical condition that results in swelling, dysfunction and / or pain of the liver, especially due to injury or infection, such as may be caused by exposure to hepatotoxic substances. Symptoms may include jaundice, fatigue, weakness, nausea, vomiting, loss of appetite, weight loss, etc. If left untreated, liver inflammation may progress to fibrosis, cirrhosis, liver failure, or liver cancer.

[0077] As used herein, "liver fibrosis", "liver fibrosis", or "fibrosis of the liver" refers to the excessive accumulation of extracellular matrix proteins in the liver, which may include collagens (I, III, and IV), FBN, undulin, elastin, laminin, hyaluronan, hepatocytes, and proteoglycans, resulting from inflammation and liver cell death. If left untreated, liver fibrosis can progress to cirrhosis, liver failure, or liver cancer.

[0078] As used herein, a "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) that is flanked by two antiparallel regions of nucleic acid that are sufficiently complementary to each other that under appropriate hybridization conditions (e.g., in a phosphate buffer solution, inside a cell), the two antiparallel regions that flanking the unpaired region hybridize to form a duplex (called a "stem").

[0079] As used herein, "modified internucleotide bond" refers to an internucleotide bond that has one or more chemical modifications compared to a reference internucleotide bond that has a phosphodiester bond. The modified nucleotide may be a non-naturally occurring bond. Typically, the modified internucleotide bond confers one or more desirable properties to the nucleic acid in which the modified internucleotide bond is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, resistance to nucleases, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0080] As used herein, "modified nucleotide" refers to a nucleotide that has one or more chemical modifications when compared to a corresponding reference nucleotide selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. The modified nucleotide may be a non-naturally occurring nucleotide. The modified nucleotide may have one or more chemical modifications, for example, in its sugar, nucleobase, and / or phosphate group. Additionally or alternatively, the modified nucleotide may have one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0081] As used herein, "nicked tetraloop structure" refers to a structure of an RNAi oligonucleotide characterized by separate sense and antisense strands, where the sense strand has a region complementary to the antisense strand, and at least one of the strands, typically the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed in at least one strand.

[0082] As used herein, "nucleoside" refers to a nucleobase-sugar combination, where the nucleobase moiety is typically a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and pyrimidines. The sugar is typically a pentose sugar, such as ribose or deoxyribose (e.g., 2'-deoxyribose).

[0083] As used herein, "nucleotide" means an organic molecule that has a nucleoside (e.g., a nucleic acid base such as adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar such as ribose or 2'-deoxyribose) and a phosphate group and can function as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0084] As used herein, "oligonucleotide" refers to a short nucleic acid molecule (e.g., less than 100 oligonucleotides in length). An oligonucleotide may be single-stranded (ss) or ds. An oligonucleotide may or may not have a double-stranded region. As a non-limiting set of examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (DsiRNA), an antisense oligonucleotide (ASO), a short siRNA, or a ss siRNA. Typically, a ds oligonucleotide is an RNAi oligonucleotide.

[0085] As used herein, "overhang" refers to terminal non-base-paired nucleotide(s) resulting from one strand or region extending beyond the end of the complementary strand with which it forms a duplex. An overhang may include one or more non-paired nucleotides extending from the duplex region at the 5' or 3' end of the ds oligonucleotide. An overhang may be a 3' or 5' overhang on the antisense or sense strand of the ds oligonucleotide.

[0086] As used herein, "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is placed at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. The 5' phosphate analog may include a phosphatase-resistant linkage. Suitable phosphate analogs include 5'-phosphonates, such as 5'-methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). An oligonucleotide may have a phosphate analog at the 4' carbon position of the sugar at the 5'-terminal nucleotide (referred to as a 4'-phosphate analog). An example of a 4'-phosphate analog is an oxymethyl phosphonate or analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., the 4-' carbon). See, for example, International Patent Application Publication No. WO2018 / 045317. Other modifications to the 5' end of oligonucleotides have been developed (see, e.g., International Patent Application No. WO2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015) Nucleic Acids Res. 43:2993-3011).

[0087] As used herein, or "SCAP-associated state", "SCAP-associated disease", or "SCAP-associated disorder" refers to a disease, disorder, or condition having increased SCAP activity and / or in which, for example, a SCAP polymorphism is present. Exemplary SCAP-associated states, diseases, or disorders include, but are not limited to, AH, ALD, CCA, cirrhosis, liver fibrosis, hepatitis, HCC, fatty liver, NAFLD, NASH, and PSC, and associated diseases, disorders, and conditions in individuals such as, for example, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, ASCVD, diabetes, and / or obesity.

[0088] As used herein, "reduced expression" or "reduced activity" refers to a reduction in the amount or level of an RNA transcript (e.g., SCAP mRNA) or a protein encoded by a gene (e.g., SCAP protein) in a cell, population of cells, sample, or subject, and / or a reduction in the amount or level of activity of the gene or protein in a cell, when compared to an appropriate reference (e.g., a reference cell, population of cells, sample, or individual). For example, the act of contacting a cell with an oligonucleotide herein (e.g., an oligonucleotide having an antisense strand having a nucleotide sequence complementary to a nucleotide sequence comprising SCAP mRNA) can result in a reduction in the amount or level of mRNA, protein, and / or activity (e.g., by degradation of SCAP mRNA by the RNAi pathway) when compared to a cell not treated with the ds oligonucleotide. Similarly, as used herein, "reducing expression" or "reducing activity" refers to an act that results in reduced expression of a gene (e.g., SCAP). Specifically, as used herein, "reduced SCAP expression" or "reduced SCAP activity" means a reduction in the amount or level of SCAP activity, e.g., SCAP mRNA, and / or SCAP protein, and / or SCAP activity, in a cell, population of cells, sample, or subject, e.g., when compared to an appropriate reference (e.g., a reference cell, population of cells, tissue, or individual).

[0089] As used herein, "region of complementarity" means a sequence of nucleotides of a nucleic acid (e.g., a ds oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions (e.g., in a phosphate buffer, inside a cell, etc.). The oligonucleotides herein include targeting sequences that have a region that is complementary to an mRNA target sequence.

[0090] As used herein, "ribonucleotide" refers to a nucleotide having as its pentose sugar a ribose with a hydroxyl group at the 2' position. A modified ribonucleotide is a ribonucleotide that has one or more modified substitutions of an atom other than the 2' position, including modifications or substitutions of the nucleobase, sugar, or phosphate group.

[0091] As used herein, "iRNA," "iRNA agent," "RNAi," "RNAi agent," and "RNA interference agent" refer to an agent, such as an RNAi oligonucleotide, that contains RNA and mediates targeted cleavage of an RNA transcript via the RNA-induced silencing complex (RISC) pathway, leading to sequence-specific degradation of mRNA via RNA interference. The agent thus regulates, inhibits, or reduces gene expression in a cell.

[0092] As used herein, "RNAi oligonucleotide" refers to either (a) a ds oligonucleotide having a sense and an antisense strand, where the antisense strand, or a portion of the antisense strand, is used to cleave a target mRNA by Argonaute 2 (Ago2) endonuclease, or (b) a ss oligonucleotide having a single-stranded antisense strand, where the antisense strand (or a portion of the antisense strand) is used to cleave a target mRNA by Ago2 endonuclease.

[0093] As used herein, a "strand" refers to a single, contiguous sequence of nucleotides linked together via internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). A strand has two free ends (e.g., a 5' end and a 3' end).

[0094] As used herein, "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine, such as a solid phase nucleic acid synthesizer) or is not derived from a natural source (e.g., a cell or organism) that normally produces nucleic acids or other molecules.

[0095] As used herein, "targeting ligand" refers to a molecule (e.g., an amino sugar, carbohydrate, cholesterol, lipid, or polypeptide) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and can be conjugated to another substance to target the other substance to the tissue or cell of interest. For example, a targeting ligand can be conjugated to the oligonucleotide herein to target the oligonucleotide to a specific tissue or cell of interest. The targeting ligand can selectively bind to a cell surface receptor. Thus, when conjugated to the oligonucleotide, the targeting ligand facilitates delivery of the oligonucleotide to a specific cell via selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex comprising the oligonucleotide, the targeting ligand, and the receptor. Additionally, the targeting ligand can be conjugated to the oligonucleotide via a linker that is cleaved after cellular internalization or during cellular internalization so that the oligonucleotide is released from the targeting ligand inside the cell.

[0096] As used herein, "tetraloop" or "teraL" refers to a loop that increases the stability of a contiguous duplex formed by hybridization of an adjacent sequence of nucleotides. The increase in stability is determined by the predicted T of the adjacent stem duplex as an average from a set of loops of equivalent length made of randomly selected sequences of nucleotides. m The melting temperature (T m For example, tetraL is a hairpin containing a duplex of at least 2 base pairs in length that can be detected by the addition of 10 mM NaHPO 4 In the present invention, a T of at least 50° C., at least 55° C., at least 56° C., at least 58° C., at least 60° C., at least 65° C., or at least 75° C. mThe tetraL can also stabilize the bp of the adjacent stem duplex by stacking interactions. In addition, interactions between nucleotides in the tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonds, and contact interactions (Cheong et al. (1990) Nature 346:680-82; Heus & Pardi (1991) Science 253:191-94). Here, the tetraL can comprise or have about 3 to about 6 nucleotides, typically about 4 to about 5 nucleotides. Thus, the tetraL can have 3, 4, 5, or 6 nucleotides, particularly 4 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). Any nucleotide can be used in tetraL, and the standard IUPAC-IUB symbols for such nucleotides can be used as described in Cornish-Bowden (1985) Nucleic Acids Res. 13:3021-30. For example, the letter "N" can be used to mean that any base can be at that position, the letter "R" can be used to indicate that A (adenine) or G (guanine) can be at that position, and "B" can be used to indicate that C (cytosine), G (guanine), or T (thymine) can be at that position. Examples of tetraLs include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al. (1990) Proc. Natl. Acad. Sci. USA 87:8467-71; Antao et al. (1991) Nucleic Acids Res. 19:5901-05).Examples of DNA tetraloops include tetraloops of the d(GNNA) family (e.g., d(GTTA)), tetraloops of the d(GNRA) family, tetraloops of the d(GNAB) family, tetraloops of the d(CNNG) family, and tetraloops of the d(TNCG) family (e.g., d(TTCG)). See, for example, Nakano et al. (2002) Biochem. 41:4281-92; and Shinji et al. (2000) Nippon Kagakkai Koen Yokoshu 78:731. Here, the tetraL can be contained within a nicked tetraL structure.

[0097] As used herein, "treat" or "treating" refers to, for example, treating an existing disease, disorder, or condition. It refers to the act of providing care to an individual in need thereof by administering a therapeutic agent (e.g., an oligonucleotide herein) to the individual for the purpose of improving the individual's health and / or well-being, or to prevent or reduce the likelihood of the occurrence of a disease, disorder, or condition. Treating can also include reducing the frequency or severity of at least one sign, symptom, or contributing factor of a disease, disorder, or condition experienced by the individual.

[0098] composition Oligonucleotide inhibitors of SCAP activity I. SCAP target sequence: The oligonucleotides herein (e.g., antisense strands of ds oligonucleotides, such as RNAi oligonucleotides) are targeted to a target sequence in the mRNA of SCAP. For example, the oligonucleotides, or portions, fragments, or strands thereof, bind to or anneal to a target sequence in the mRNA of SCAP, thereby inhibiting SCAP activity. In some embodiments, the oligonucleotides are targeted to a SCAP target sequence to inhibit SCAP activity in vivo. In some embodiments, the amount or degree of inhibition of SCAP activity by oligonucleotides targeted to a SCAP target sequence correlates with the efficacy of the oligonucleotide. In some embodiments, the amount or degree of inhibition of SCAP activity by oligonucleotides targeted to a SCAP target sequence correlates with the amount or degree of therapeutic effect in individuals treated with the oligonucleotide who have or are suspected of having a disease, disorder, or condition associated with SCAP activity.

[0099] By examining and analyzing the nucleotide sequence of SCAP mRNA, including mRNA from multiple different species (e.g., human, mouse, and / or monkey, see, e.g., Example 2), as well as the results of in vitro and in vivo testing (see, e.g., Examples 3 and 4), it is shown herein that certain nucleotide sequences of SCAP mRNA are more amenable to oligonucleotide-based inhibition of SCAP activity than other nucleotide sequences, and thus are useful as target sequences for the oligonucleotides herein. In some embodiments, the sense strand of the oligonucleotides herein (e.g., ds oligonucleotides such as RNAi oligonucleotides, e.g., in Table 3) comprises a SCAP target sequence. In some examples, a portion or region of the sense strand of the RNAi oligonucleotides herein (e.g., in Table 3) comprises a SCAP target sequence. In some embodiments, the SCAP target sequence comprises or consists of any one of SEQ ID NOs: 777-783 or any one of the odd numbers of SEQ ID NOs: 785-1168 (in particular, any one of SEQ ID NOs: 915, 923, 997, 1049, 1097, 1109, and 1137).

[0100] II. SCAP mRNA targeting sequence: In some embodiments, the oligonucleotides herein (e.g., the antisense strand of a ds oligonucleotide, such as an RNAi oligonucleotide) have a region of complementarity to SCAP mRNA (e.g., within the target sequence of SCAP mRNA) to target SCAP mRNA in cells and inhibit SCAP activity. In some embodiments, the oligonucleotides include a SCAP targeting sequence (e.g., the antisense strand of a ds oligonucleotide) with a region of complementarity that binds or anneals to the SCAP mRNA target sequence by complementary (Watson-Crick) base pairing. The region of complementarity is of appropriate length and base content to allow the oligonucleotide (or a strand thereof) to bind or anneal to SCAP mRNA to inhibit expression of SCAP mRNA. In some embodiments, the targeting sequence or region of complementarity is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides in length. Alternatively, the targeting sequence or region of complementarity is about 12-30 (e.g., 12-30, 12-22, 15-25, 17-21, 18-27, 19-27, or 15-30) nucleotides in length. Alternatively, the targeting sequence or region of complementarity is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In certain embodiments, the targeting sequence or region of complementarity is 18 nucleotides in length. In certain embodiments, the targeting sequence or region of complementarity is 19 nucleotides in length. In certain embodiments, the targeting sequence or region of complementarity is 20 nucleotides in length. In certain embodiments, the targeting sequence or region of complementarity is 21 nucleotides in length.In certain embodiments, the targeting sequence or region of complementarity is 22 nucleotides in length. In certain embodiments, the targeting sequence or region of complementarity is 23 nucleotides in length. In certain embodiments, the targeting sequence or region of complementarity is 24 nucleotides in length.

[0101] In some embodiments, the oligonucleotides herein comprise a targeting sequence or region of complementarity that is fully complementary to the SCAP mRNA targeting sequence (e.g., the antisense strand of a ds oligonucleotide). In some embodiments, the targeting sequence or region of complementarity is partially complementary to the SCAP mRNA targeting sequence. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is fully complementary to any one of SEQ ID NOs: 777-783. In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is partially complementary to any one of SEQ ID NOs: 777-783.

[0102] Alternatively, in some embodiments, the oligonucleotides herein comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising the mRNA of SCAP, the contiguous sequence of nucleotides being about 12 to about 30 nucleotides in length (e.g., 12-30, 12-28, 12-26, 12-24, 12-20, 12-18, 12-16, 14-22, 16-20, 18-20, or 18-19 nucleotides in length). In some embodiments, the oligonucleotides comprise a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising the mRNA of SCAP, the contiguous sequence of nucleotides being 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising the SCAP mRNA, wherein the contiguous sequence of nucleotides is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides comprising the SCAP mRNA, wherein the contiguous sequence of nucleotides is 20 nucleotides in length. In other embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides of any one of SEQ ID NOs: 777-783, wherein optionally the contiguous sequence of nucleotides is 19 nucleotides in length.

[0103] With respect to the targeting sequence or region of complementarity of the oligonucleotide herein, it is complementary to consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 777-783 and spans the entire length of the antisense strand. In some embodiments, the region of complementarity of the oligonucleotide is complementary to consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 777-783 and spans a portion of the entire length of the antisense strand. In some further embodiments, the oligonucleotide comprises a region of complementarity (e.g., on the antisense strand of a ds oligonucleotide) that is at least partially (e.g., completely) complementary to a consecutive stretch of nucleotides spanning nucleotides 1-20, 1-19, 1-18, etc. of the sequence set forth in any one of SEQ ID NOs: 777-783.

[0104] Alternatively, the oligonucleotide comprises a targeting sequence or region of complementarity having one or more base pair (bp) mismatches with the corresponding SCAP mRNA target sequence. In some embodiments, the targeting sequence or region of complementarity has up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, etc. mismatches with the corresponding SCAP target sequence, provided that the ability of the targeting sequence or region of complementarity to bind or anneal to SCAP mRNA under suitable hybridization conditions and / or the ability of the oligonucleotide to reduce or inhibit SCAP activity is maintained. In other words, the targeting sequence or region of complementarity has no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches with the corresponding SCAP target sequence, provided that the ability of the targeting sequence or region of complementarity to bind or anneal to SCAP mRNA under suitable hybridization conditions and / or the ability of the oligonucleotide to reduce or inhibit SCAP activity is maintained. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity with one mismatch to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity with two mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity with three mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity with four mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide comprises a target sequence or region of complementarity with five mismatches to the corresponding target sequence. In other embodiments, the oligonucleotide comprises a target sequence or region of complementarity with more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) to the corresponding target sequence, where at least two (e.g., all) of the mismatches are located contiguously (e.g., 2, 3, 4, 5 or more mismatches contiguous) or the mismatches are interspersed anywhere throughout the targeting sequence or region of complementarity.In other embodiments, the oligonucleotide comprises a target sequence or region of complementarity with more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, where at least two (e.g., all) of the mismatches are positioned contiguous (e.g., 2, 3, 4, 5 or more mismatches contiguous) or where at least one or more unmatched bp are located between the mismatches, or a combination thereof.

[0105] III. Oligonucleotide Types: A variety of oligonucleotide types and / or structures are useful for targeting SCAP mRNA, including but not limited to RNAi oligonucleotides, ASOs, miRNAs, and the like. Any of the oligonucleotide types described herein or elsewhere are intended to be used as a framework for incorporating targeting sequences herein for the purpose of inhibiting SCAP activity. In some embodiments, the oligonucleotides herein inhibit SCAP activity by engaging with the RNAi pathway upstream or downstream of Dicer involvement. For example, RNAi oligonucleotides have been developed with each strand having a size of 19-25 nucleotides and at least one 3'-overhang of 1-5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to produce active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further research has produced extended ds oligonucleotides, where at least one end of at least one strand extends beyond the targeting region of the duplex, with one strand comprising a structure with a thermodynamically stable tetraL (see, for example, U.S. Pat. Nos. 8,513,207 and 8,927,705, and International Patent Publication No. WO2010 / 033225). Such structures include ss extensions (on one or both sides of the molecule) and ds extensions.

[0106] The oligonucleotides herein are involved in the RNAi pathway downstream of Dicer involvement (e.g., Dicer cleavage). In some embodiments, the oligonucleotides have an overhang (e.g., 1, 2, or 3 nucleotides long) at the 3' end of the sense strand. In some embodiments, the oligonucleotides (e.g., siRNAs) comprise a 21 nucleotide antisense strand that is antisense to the target mRNA (e.g., SCAP mRNA) and a complementary sense strand, both strands annealing to form a 19 bp duplex and a 2 nucleotide overhang at one or both 3' ends. Longer oligonucleotide designs are also contemplated, including oligonucleotides with a 23 nucleotide long antisense strand and a 21 nucleotide long sense strand, with a blunt end on the right side of the molecule (3' end of sense strand / 5' end of antisense strand) and a 2 nucleotide 3' antisense strand overhang on the left side of the molecule (5' end of sense strand / 3' end of antisense strand). Such molecules have a 21 bp duplex region. See, e.g., U.S. Patent Nos. 9,012,138; 9,012,621; and 9,193,753.

[0107] The oligonucleotides herein comprise a sense strand and an antisense strand, both ranging from about 17 to about 26 (e.g., 17-26, 20-25, or 21-23) nucleotides in length. In some embodiments, the oligonucleotides comprise a sense strand and an antisense strand, both ranging from about 19 to about 22 nucleotides in length. In some embodiments, the sense and antisense strands are of equal length. In some embodiments, the oligonucleotides comprise a sense strand and an antisense strand, and there is a 3' overhang on either the sense strand or the antisense strand, or on both the sense and antisense strands. In some embodiments, for oligonucleotides having a sense strand and an antisense strand, both ranging from about 21 to about 23 nucleotides in length, the 3' overhang on the sense strand, the antisense strand, or on both the sense and antisense strands is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has an antisense strand 22 nucleotides long and a sense strand 20 nucleotides long, with a blunt end on the right side of the molecule (3' end of sense strand / 5' end of antisense strand) and a 2 nucleotide 3' antisense strand overhang on the left side of the molecule (5' end of sense strand / 3' end of antisense strand).Such molecules have a 20 bp double-stranded region.

[0108] Other oligonucleotide designs for use herein include 16-mer siRNAs (see, e.g., "Nucleic Acids in Chemistry & Biology," Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., those with stems of 19 bp or shorter; see, e.g., Moore et al. (2010) Methods Mol. Biol. 629:141-58), blunt siRNAs (e.g., those 19 bp in length; see, e.g., Kraynack & Baker (2006) RNA 12:163-76), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al. (2008) Nat. Biotechnol. 26:1379-82), asymmetric short duplex siRNAs (see, e.g., Chang et al. (2008) Nat. Biotechnol. 26:1379-82), and the like. al. (2009) Mol. Ther. 17:725-32), forked siRNAs (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-98), ss siRNAs (see, e.g., Elsner (2012) Nat. Biotechnol. 30:1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. (2007) J. Am. Chem. Soc. 129:15108-09), and small internal segmented interfering RNAs (sisiRNAs; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35:5886-97). Further non-limiting examples of oligonucleotide structures that can be used herein to reduce or inhibit SCAP activity are miRNAs, shRNAs, and short siRNAs (see, e.g., Hamilton et al. (2002) EMBO J. 21:4671-79; see also U.S. Patent No. 7,659,389).

[0109] Alternatively, the oligonucleotide herein is ss. Such structures include, but are not limited to, ss RNAi molecules. Recent efforts have demonstrated the activity of ss RNAi molecules (see, for example, Matsui et al. (2016) Mol. Ther. 24:946-55). In some embodiments, the oligonucleotide is an ASO. An ASO is an ss oligonucleotide having a nucleobase sequence that, when written or depicted in a 5' to 3' direction, contains the reverse complement of a targeting segment of a particular nucleic acid, and is appropriately modified (e.g., as a gapmer) to induce RNaseH-mediated cleavage of that target RNA in a cell, or inhibit translation of a target mRNA in a cell (e.g., as a mixmer). ASOs for use herein are modified in any suitable manner known in the art, including, for example, those shown in U.S. Pat. No. 9,567,587 (including, for example, modifications of the length of the nucleobase, the sugar moiety (pyrimidine, purine), and the heterocyclic moiety of the nucleobase). Moreover, ASOs have been used for decades to reduce the expression of specific target genes (see, e.g., Bennett et al. (2017) Annu. Rev. Pharmacol. 57:81-105).

[0110] IV. ds RNAi oligonucleotides: ds oligonucleotides for targeting SCAP mRNA and inhibiting SCAP activity (e.g., via the RNAi pathway) comprise a sense strand and an antisense strand. In some embodiments, the antisense strand and the sense strand are separate strands and are not covalently linked. In some embodiments, the antisense strand and the sense strand are covalently linked.

[0111] In some embodiments, the sense strand comprises a first region (R1) and a second region (R2), R2 comprises a first subregion (S1), triL or L, and a second subregion (S2), where triL or L is located between S1 and S2, and S1 and S2 form a second duplex (D2). D2 has a variety of lengths. In some embodiments, D2 is about 1 to about 6 bp in length. In other embodiments, D2 is 2 to 6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, or 4 to 5 bp in length. In other embodiments, D2 is 1, 2, 3, 4, 5, or 6 bp in length. In certain embodiments, D2 is 6 bp in length.

[0112] In some embodiments, R1 of the sense strand and the antisense strand form a first duplex (D1). In some embodiments, D1 is at least 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In other embodiments, D1 is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30, or 21 to 30 nucleotides in length). In other embodiments, D1 is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In other embodiments, D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, D1 is 20 nucleotides in length. In some embodiments, D1 does not span the entire length of the sense strand and / or the antisense strand. In other embodiments, D1 spans the entire length of either the sense strand or the antisense strand, or both. In some embodiments, D1 spans the entire length of both the sense strand and the antisense strand.

[0113] In certain embodiments, the present disclosure describes RNAi oligonucleotides for reducing or inhibiting SCAP activity, comprising a sense strand that includes or alternatively consists of a sequence set forth in Table 3 (e.g., any one of the odd numbers of SEQ ID NOs: 9-392), particularly SEQ ID NOs: 139, 147, 221, 273, 321, 333, and 361.

[0114] In certain embodiments, the present disclosure describes RNAi oligonucleotides for reducing or inhibiting SCAP activity, comprising an antisense strand comprising or alternatively consisting of a sequence set forth in Table 3 (e.g., any one of the even numbers of SEQ ID NOs: 9-392), particularly SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362.

[0115] In certain other embodiments, the RNAi oligonucleotide comprises a sense strand that comprises, or alternatively consists of, the nucleotide sequence of any one of SEQ ID NOs: 139, 147, 221, 273, 321, 333, and 361, and an antisense strand that comprises, or alternatively consists of, the nucleotide sequence of any one of SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362.

[0116] In certain embodiments, the sense and antisense strands of the RNAi oligonucleotide are each selected from the following: (a) SEQ ID NOs: 139 and 140; (b) SEQ ID NOs: 147 and 148; (c) SEQ ID NOs: 221 and 222; (d) SEQ ID NOs: 273 and 274; (e) SEQ ID NOs: 321 and 322; (f) SEQ ID NOs: 333 and 334; (g) SEQ ID NOs: 361 and 362.

[0117] In certain embodiments, the RNAi oligonucleotide comprises a sense strand that comprises, or alternatively consists of, the nucleotide sequence of SEQ ID NO: 147 or 333, and an antisense strand that comprises, or alternatively consists of, the nucleotide sequence of any one of SEQ ID NO: 148 or 334. Alternatively, the sense strand is SEQ ID NO: 147 and the antisense strand is SEQ ID NO: 148. Alternatively, the sense strand is SEQ ID NO: 333 and the antisense strand is SEQ ID NO: 334.

[0118] In some embodiments, the RNAi oligonucleotide comprises a sense strand that comprises or alternatively consists of a nucleotide sequence set forth in Table 4 (e.g., any one of the odd numbers of SEQ ID NOs: 393-776), particularly SEQ ID NOs: 523, 531, 605, 657, 705, 717, and 745.

[0119] In certain embodiments, the RNAi oligonucleotide comprises an antisense strand that comprises or alternatively consists of a nucleotide sequence set forth in Table 4 (e.g., any one of the even numbers of SEQ ID NOs: 393 to 776), particularly SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746.

[0120] In certain other embodiments, the RNAi oligonucleotide comprises a sense strand that comprises, or alternatively consists of, the nucleotide sequence of any one of SEQ ID NOs: 523, 531, 605, 657, 705, 717, and 745, and an antisense strand that comprises, or alternatively consists of, the nucleotide sequence of any one of SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746.

[0121] In certain embodiments, the sense and antisense strands of the RNAi oligonucleotide are each selected from the following: (a') SEQ ID NOs: 523 and 524; (b') SEQ ID NOs: 531 and 532; (c') SEQ ID NOs: 605 and 606; (d') SEQ ID NOs: 657 and 658; (e') SEQ ID NOs: 705 and 706; (f') SEQ ID NOs: 717 and 718; (g') SEQ ID NOs: 745 and 746.

[0122] In certain further embodiments, the RNAi oligonucleotide comprises a sense strand that comprises, or alternatively consists of, the nucleotide sequence of SEQ ID NO: 531 or 717, and an antisense strand that comprises, or alternatively consists of, SEQ ID NO: 532 or 718. Alternatively, the sense strand is SEQ ID NO: 531 and the antisense strand is SEQ ID NO: 532. Alternatively, the sense strand is SEQ ID NO: 717 and the antisense strand is SEQ ID NO: 718.

[0123] Those skilled in the art will appreciate that in some embodiments, reference is made to the sequences set forth in the sequence listing to describe the structure of an oligonucleotide (e.g., a ds oligonucleotide, such as an RNAi oligonucleotide) or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid retains essentially the same or similar complementary properties as the identified sequence, but has one or more alternative nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications compared to the identified sequence.

[0124] In some embodiments, the oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) comprise a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, which upon action of the Dicer enzyme produces an antisense strand that is incorporated into mature RISC. In other embodiments, the sense strand of the ds oligonucleotide is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides). In other embodiments, the sense strand of the ds oligonucleotide is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).

[0125] In some embodiments, the oligonucleotide has one 5' end that is thermodynamically unstable compared to the other 5' end. In some embodiments, the oligonucleotide is asymmetric and comprises a blunt end at the 3' end of the sense strand and a 3' overhang at the 3' end of the antisense strand. In some embodiments, the 3' overhang on the antisense strand is about 1 to about 8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides in length). Typically, ds oligonucleotides for RNAi have a 2 nucleotide overhang at the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang having a length of about 1 to about 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides. However, in other embodiments, the overhang is a 5' overhang comprising a length of about 1 to about 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides.

[0126] In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand are modified. In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand are complementary to the target mRNA (e.g., SCAP mRNA). In other embodiments, the two terminal nucleotides at the 3' end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand of the nucleotide herein are not paired. In some embodiments, the two terminal nucleotides at the 3' end of each of the oligonucleotides in the nicked tetraL structure are GG. Typically, one or both of the two terminal GG nucleotides at the 3' end of each of the ds oligonucleotides are not complementary to the target mRNA.

[0127] In some embodiments, there is one or more (e.g., 1, 2, 3, 4, 5) mismatch(es) between the sense strand and the antisense strand. When there is more than one mismatch between the sense strand and the antisense strand, they may be arranged consecutively (e.g., 2, 3, or more consecutively) or may be interspersed throughout the region of complementarity. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In certain embodiments, two mismatches are incorporated into the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of a segment at the 3' end of the sense strand of an oligonucleotide improves or increases the potency of a ds oligonucleotide.

[0128] In some embodiments, there are one or more (e.g., 1, 2, 3, 4, 5) mismatch(s) between the sense strand and the antisense strand comprising the oligonucleotide herein (e.g., a ds oligonucleotide such as an RNAi oligonucleotide). When there is more than one mismatch between the sense strand and the antisense strand, they may be arranged consecutively (e.g., 2, 3, or more consecutively) or may be interspersed throughout the region of complementarity. In some embodiments, the 3' end of the sense strand comprises one or more mismatches. In some embodiments, two mismatches are incorporated into the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of a segment at the 3' end of the sense strand of the oligonucleotide herein improves or increases the potency of the oligonucleotide. In some embodiments, the sense and antisense strands of the oligonucleotide herein are selected from Table 3, optionally including: (a) SEQ ID NOs: 139 and 140; (b) SEQ ID NOs: 147 and 148; (c) SEQ ID NOs: 221 and 222; (d) SEQ ID NOs: 273 and 274; (e) SEQ ID NOs: 321 and 322; (f) SEQ ID NOs: 333 and 334; (g) SEQ ID NOs: 361 and 362 wherein there are one or more (e.g., 1, 2, 3, 4, 5) mismatch(es) between the sense and antisense strands.

[0129] A. Sense Strand: The oligonucleotides (e.g., ds oligonucleotides, such as RNAi oligonucleotides) herein include a sense strand sequence that includes a sequence set forth in the sense strand of Table 3 or Table 4. In some embodiments, the oligonucleotides include a sense strand having at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 139, 147, 221, 273, 321, 333, and 361, or a sense strand having the nucleotide sequence of any one of SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362.

[0130] Additionally, the oligonucleotide may comprise a sense strand up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide may have a sense strand at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). Alternatively, the oligonucleotides can have a sense strand ranging in length from about 12 to about 40 nucleotides (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-21, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In certain embodiments, the oligonucleotides can have a sense strand length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

[0131] In some embodiments, the sense strand comprises a stem-loop structure at the 3' end. In some embodiments, the sense strand comprises a stem-loop structure at the 5' end. In some embodiments, the stem-loop is formed by intrastrand base pairing. In further embodiments, the stem is a duplex about 2 bp, about 3 bp, about 4 bp, about 5 bp, about 6 bp, about 7 bp, about 8 bp, about 9 bp, about 10 bp, about 11 bp, about 12 bp, about 13 bp, or about 14 bp in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 7 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 8 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 9 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 10 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 11 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 12 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 13 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex that is 14 nucleotides in length.

[0132] In some embodiments, the stem loop provides protection to the oligonucleotide from degradation (e.g., enzymatic degradation), and / or facilitates or enhances targeting and / or delivery to a target cell, tissue, or organ (e.g., liver). For example, the loop of the stem loop provides a nucleotide with one or more modifications that facilitate, enhance, or increase targeting to a target mRNA (e.g., SCAP mRNA), inhibition of target gene expression (e.g., SCAP activity), and / or delivery to a target cell, tissue, or organ (e.g., liver). In some embodiments, the stem loop itself or the modification(s) to the stem loop do not substantially affect the intrinsic gene expression inhibitory activity of the oligonucleotide, but facilitate, improve, or increase stability (e.g., providing protection against degradation) and / or delivery of the oligonucleotide to a target cell, tissue, or organ (e.g., liver). In certain embodiments, the oligonucleotide comprises a sense strand that comprises (e.g., at its 3' end) a stem loop designated as S1-L-S2, where S1 is complementary to S2, and L forms a ss loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, L is 3 nucleotides in length (referred to herein as a "tri-loop" or "triL"). In some embodiments, L is 4 nucleotides in length (referred to herein as a "tetra-loop" or "tetraL"). In some embodiments, L is 5 nucleotides in length. In some embodiments, L is 6 nucleotides in length. In some embodiments, L is 7 nucleotides in length. In some embodiments, L is 8 nucleotides in length. In some embodiments, L is 9 nucleotides in length. In some embodiments, L is 10 nucleotides in length. In some embodiments, L is 4 nucleotides in length. FIG. 1 depicts a non-limiting example of such an oligonucleotide.In some embodiments, L of the stem loop having the structure S1-L-S2 described above is tetraL (e.g., in a nicked tetraL structure). In some embodiments, tetraL comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, delivery ligands, and combinations thereof. In certain embodiments, tetraL comprises the sequence 5'-GAAA-3'. In other particular embodiments, the stem loop comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 784).

[0133] In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region that is complementary to a contiguous sequence of nucleotides in any one of the odd SEQ ID NOs: 785-1168, particularly any one of SEQ ID NOs: 915, 923, 997, 1049, 1097, 1109, and 1137, and the oligonucleotide comprises a sense strand that comprises (e.g., at its 3' end) a stem-loop depicted as S1-L-S2, where S1 is complementary to S2 and L forms a single-stranded loop between S1 and S2 up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). In some embodiments, the oligonucleotide comprises a targeting sequence or region of complementarity that is complementary to a contiguous sequence of nucleotides in any one of the odd SEQ ID NOs: 785-1168, particularly any one of SEQ ID NOs: 915, 923, 997, 1049, 1097, 1109, and 1137, and the oligonucleotide comprises a sense strand that comprises (e.g., at its 3' end) a stem-loop designated as S1-L-S2, where S1 is complementary to S2 and L forms a single-stranded loop between S1 and S2 that is 4 nucleotides in length.

[0134] In some embodiments, L of the stem-loop having the structure S1-L-S2 described herein is triL. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region, and triL, that is complementary to a contiguous sequence of nucleotides in any one of the odd numbers of SEQ ID NOs: 785-1168, particularly any one of SEQ ID NOs: 915, 923, 997, 1049, 1097, 1109, and 1137. In some embodiments, triL comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands), and combinations thereof.

[0135] In some embodiments, L of the stem-loop having the structure S1-L-S2 described herein is tetraL. In some embodiments, the oligonucleotide comprises a targeting sequence or complementary region, and tetraL, that is complementary to a contiguous sequence of nucleotides in any one of the odd numbers of SEQ ID NOs: 785-1168, particularly any one of SEQ ID NOs: 915, 923, 997, 1049, 1097, 1109, and 1137. In some embodiments, tetraL comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands), and combinations thereof.

[0136] B. Antisense Strand: Oligonucleotides (e.g., ds oligonucleotides, such as RNAi oligonucleotides) herein include an antisense strand that includes a sequence set forth in the antisense strand of Table 3 (unmodified) or Table 4 (modified). In some embodiments, the oligonucleotide includes an antisense strand having at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 139, 147, 221, 273, 321, 333, and 361, or an antisense strand having the nucleotide sequence of any one of SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362.

[0137] Additionally, the oligonucleotide may comprise an antisense strand up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide comprises an antisense strand at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, the oligonucleotide comprises an antisense strand ranging in length from about 12 to about 40 nucleotides (e.g., 12-40, 12-36, 12-32, 12-28, 15-40, 15-36, 15-32, 15-28, 17-22, 17-25, 19-27, 19-30, 20-40, 22-40, 25-40, or 32-40). In some embodiments, the oligonucleotide comprises an antisense strand ranging in length from about 12 to about 40 nucleotides. In some embodiments, the antisense strand of any one of the oligonucleotides disclosed herein is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense strand that is 22 nucleotides in length.

[0138] In some embodiments, the oligonucleotide comprises an antisense strand comprising or consisting of a sequence listed in Table 3 (e.g., any one of the odd numbers of SEQ ID NOs: 9-392). In some embodiments, the oligonucleotide herein comprises an antisense strand comprising at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence listed in Table 3 (e.g., any one of the even numbers of SEQ ID NOs: 9-392). In some embodiments, the oligonucleotide disclosed herein for targeting SCAP comprises an antisense strand comprising or consisting of any one of the sequences listed in Table 3 (e.g., any one of the even numbers of SEQ ID NOs: 9-392), in particular SEQ ID NOs: 140, 148, 222, 274, 322, 334, and 362. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand comprising at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in Table 4 (e.g., any one of even numbers of SEQ ID NOs: 393-776). In some embodiments, the oligonucleotides disclosed herein for targeting SCAP comprise an antisense strand comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746. In some embodiments, the oligonucleotides herein comprise an antisense strand that comprises at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746.

[0139] C. Duplex Length: The oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) include a duplex formed between a sense strand and an antisense strand, the duplex being at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21) nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand ranges from about 12 to about 30 nucleotides in length (e.g., 12-30, 12-27, 12-22, 15-25, 18-30, 18-22, 18-25, 18-27, 18-30, 19-30, or 21-30 nucleotides in length). In some embodiments, the duplex formed between the sense strand and the antisense strand is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 12 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 13 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 14 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 15 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 16 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 17 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 18 nucleotides in length.In some embodiments, the duplex formed between the sense strand and the antisense strand is 19 nucleotides in length.In some embodiments, the duplex formed between the sense strand and the antisense strand is 20 nucleotides in length.In some embodiments, the duplex formed between the sense strand and the antisense strand is 21 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 22 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 23 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 24 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 25 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 26 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 27 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 28 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 29 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand is 30 nucleotides in length. In some embodiments, the duplex formed between the sense strand and the antisense strand does not span the entire length of the sense strand and / or the antisense strand. In some embodiments, the duplex formed between the sense strand and the antisense strand spans the entire length of either the sense strand or the antisense strand. In some embodiments, the duplex formed between the sense strand and the antisense strand spans the entire length of both the sense strand and the antisense strand.

[0140] D. Oligonucleotide Termination: The oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) comprise a sense strand and an antisense strand, and the termini of either or both strands comprise blunt ends. In some embodiments, the oligonucleotides comprise a sense strand and an antisense strand, which are separate strands forming an asymmetric duplex region with an overhang at the 3' end of the antisense strand. In some embodiments, the oligonucleotides comprise a sense strand and an antisense strand, and the termini of either or both strands comprise an overhang comprising one or more nucleotides. In some embodiments, the one or more nucleotides comprising the overhang are unpaired nucleotides. In some embodiments, the oligonucleotides comprise a sense strand and an antisense strand, and the 3' end of the sense strand and the 5' end of the antisense strand comprise blunt ends. In some embodiments, the oligonucleotides comprise a sense strand and an antisense strand, and the 5' end of the sense strand and the 3' end of the antisense strand comprise blunt ends.

[0141] In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, and the 3' end of either or both strands comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, and the sense strand comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, and the antisense strand comprises a 3' overhang comprising one or more nucleotides. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, and both the sense strand and the antisense strand comprise a 3' overhang comprising one or more nucleotides.

[0142] In some embodiments, the 3' overhang is about 1 to about 20 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length). In some embodiments, the 3' overhang is about 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides in length. In some embodiments, the 3' overhang is 1 nucleotide in length. In some embodiments, the 3' overhang is 2 nucleotides in length. In some embodiments, the 3' overhang is 3 nucleotides long. In some embodiments, the 3' overhang is 4 nucleotides long. In some embodiments, the 3' overhang is 5 nucleotides long. In some embodiments, the 3' overhang is 6 nucleotides long. In some embodiments, the 3' overhang is 7 nucleotides long. In some embodiments, the 3' overhang is 8 nucleotides long. In some embodiments, the 3' overhang is 9 nucleotides long. In some embodiments, the 3' overhang is 10 nucleotides long. In some embodiments, the 3' overhang is 11 nucleotides long. In some embodiments, the 3' overhang is 12 nucleotides long. In some embodiments, the 3' overhang is 13 nucleotides long. In some embodiments, the 3' overhang is 14 nucleotides long. In some embodiments, the 3' overhang is 15 nucleotides long. In some embodiments, the 3' overhang is 16 nucleotides long. In some embodiments, the 3' overhang is 17 nucleotides long. In some embodiments, the 3' overhang is 18 nucleotides long. In some embodiments, the 3' overhang is 19 nucleotides in length. In some embodiments, the 3' overhang is 20 nucleotides in length.

[0143] In certain embodiments, the oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 3' overhang.

[0144] V. Oligonucleotide Modifications: The oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) contain at least one modification. Oligonucleotides can be modified in a variety of ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other properties relevant for therapeutic or research applications.

[0145] In some embodiments, the modification is a modified sugar. In some embodiments, the modification is a 5' terminal phosphate group. In some embodiments, the modification is a modified internucleotide linkage. In some embodiments, the modification is a modified base. In some embodiments, the oligonucleotide comprises any one of the modifications described herein or any combination thereof. For example, in some embodiments, the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base. In some embodiments, the sense and antisense strands of the oligonucleotide are selected from Table 3, optionally including: (a) SEQ ID NOs: 139 and 140; (b) SEQ ID NOs: 147 and 148; (c) SEQ ID NOs: 221 and 222; (d) SEQ ID NOs: 273 and 274; (e) SEQ ID NOs: 321 and 322; (f) SEQ ID NOs: 333 and 334; (g) SEQ ID NOs: 361 and 362 and wherein the oligonucleotide comprises at least one modified sugar, a 5' terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base.

[0146] In another embodiment, the oligonucleotide comprises a sense strand and an antisense strand having a modification pattern according to the following: Sense strand: 5'-mX-S-mX-mX-mX-mX-mX-mX-fX-fX-fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-[ademX-GalNAc]-[ademX-GalNAc]-[ademX-GalNAc]-mX-mX-mX-mX-mX-3', hybridizes to:

[0147] antisense strand: 5'-[MePhosphonate-4O-mX]-S-fX-S-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-S-mX-S-mX-3', where mX = 2'-OMe-modified nucleotide, fX = 2'-F-modified nucleotide, -S- = phosphorothioate linkage, - = phosphodiester linkage, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and ademX-GalNAc = GalNAc linked to a nucleotide, wherein the sense strand and the antisense strand are selected from Table 3, and optionally (a) SEQ ID NOs: 139 and 140; (b) SEQ ID NOs: 147 and 148; (c) SEQ ID NOs: 221 and 222; (d) SEQ ID NOs: 273 and 274; (e) SEQ ID NOs: 321 and 322; (f) SEQ ID NOs: 333 and 334; (g) comprising a nucleotide sequence selected from SEQ ID NOs: 361 and 362.

[0148] A. Sugar Modifications: Modified sugars (also referred to herein as sugar analogs) include modified deoxyribose or ribose moieties, e.g., where one or more modifications occur at the 2', 3', 4', and / or 5' carbons of the sugar. Modified sugars also include locked nucleic acids ("LNAs", see e.g., Koshkin et al. (1998) Tetrahedron 54:3607-30), unlocked nucleic acids ("UNAs", see e.g., Snead et al. (2013) Mol. Ther-Nuc. Acids 2:e103), and bridged nucleic acids ("BNAs"; see e.g., Imanishi & Obika (2002) Chem. Commun. 16:1653-59).

[0149] In some embodiments, the nucleotide modification in the sugar is a 2'-modification, such as, for example, 2'-O-propargyl, 2'-O-propylamine, 2'-amino, 2'-ethyl, 2'-F, 2'-aminoethyl (EA), 2'-OMe, 2'-MOE, 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), or 2'-FANA. In certain embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In some embodiments, the modification in the sugar includes a modification of the sugar ring, which can include a modification of one or more carbons of the sugar ring. For example, the modification in the sugar is the 2'-oxygen of the sugar attached to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen attached to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modification is an acyclic sugar that lacks a bond between the 2' and 3' carbons. In other embodiments, the modification is a thiol group, for example at the 4' position of the sugar.

[0150] The oligonucleotides herein comprise at least one (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more) modified nucleotide. In some embodiments, the sense strand comprises at least one (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more) modified nucleotide. In some embodiments, the antisense strand comprises at least one (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more) modified nucleotide.

[0151] In certain embodiments, all nucleotides of the sense strand, except tetraL, are modified. Similarly, all nucleotides of the antisense strand are modified. In some embodiments, all nucleotides of the oligonucleotide (i.e., nucleotides paired in the sense and antisense strands) are modified. As described above, and in some embodiments, the modified nucleotides are 2'-modified (e.g., 2'-F, 2'-OMe, 2'-MOE, and / or 2'-FANA). In certain embodiments, the modified nucleotides are 2'-modified, such as, for example, 2'-F or 2'-OMe.

[0152] In some embodiments, the oligonucleotide comprises a sense strand, and about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand, and about 18-23% (e.g., 18%, 19%, 20%, 21%, 22%, or 23%) of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, the oligonucleotide comprises a sense strand, and about 38-43% (e.g., 38%, 39%, 40%, 41%, 42%, or 43%) of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, about 11% of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, about 22% of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, about 40% of the nucleotides of the sense strand comprise a 2'-F modification. In some embodiments, the oligonucleotide comprises an antisense strand, and about 25% to about 35% (e.g., 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%) of the nucleotides of the antisense strand comprise 2'-F modifications. In some embodiments, about 32% of the nucleotides of the antisense strand comprise 2'-F modifications. In some embodiments, the oligonucleotide comprises about 15% to about 25% (e.g., 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%) of its nucleotides comprise 2'-F modifications. In some embodiments, the oligonucleotide comprises 35%-45% (e.g., 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%) of its nucleotides comprising a 2'-F modification. In some embodiments, about 19% of the nucleotides in the oligonucleotide comprise a 2'-F modification. In some embodiments, about 29% of the nucleotides in the oligonucleotide comprise a 2'-F modification. In some embodiments, about 40% of the nucleotides in the oligonucleotide comprise a 2'-F modification.

[0153] Additionally, oligonucleotides herein may have different modification patterns. In some embodiments, a modified oligonucleotide comprises a sense strand sequence having a modification pattern as set forth in Table 4 (and FIG. 1) and an antisense strand having a modification pattern as set forth in Table 4. In some embodiments, one or more of positions 8, 9, 10, or 11 of the sense strand are modified with 2'-F. In other embodiments, the sugar moieties of each of the nucleotides at positions 1-7, 12-27, and 31-36 of the sense strand are modified with 2'-OMe. In certain embodiments, positions 8-11 of the sense strand are modified with 2'-F and positions 1-7, 12-27, and 31-36 are modified with 2'-OMe.

[0154] In more particular embodiments, the sense strand includes 2'-F modified nucleotides at positions 8-11, 2'-OMe modified nucleotides at positions 1-7, 12-27, and 31-36, GalNAc conjugated nucleotides at positions 28, 29, and 30, and a phosphorothioate linkage between positions 1 and 2.

[0155] In some embodiments, the antisense strand comprises one or more nucleotides at positions 2-5, 7, 10, and 14 that are 2'-F modified and one or more nucleotides at positions 1, 6, 8-9, 11-13, and 15-22 that are 2'-OMe modified. Certain embodiments disclose oligonucleotides having an antisense strand that comprises 2'-F modified nucleotides at positions 2-5, 7, 10, and 14 and 2'-OMe modified nucleotides at positions 1, 6, 8-9, 11-13, and 15-22.

[0156] In certain embodiments, the antisense strand contains 2'-F modified nucleotides at positions 2-5, 7, 10, and 14, 2'-OMe at positions 1, 6, 8-9, 11-13, and 15-22, and phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22.

[0157] B. 5'-Terminal Phosphate: A 5'-terminal phosphate group can be used to enhance the interaction of the oligonucleotides herein with Ago2. However, oligonucleotides with a 5'-terminal phosphate group may be susceptible to degradation by phosphatases or other enzymes, potentially limiting their bioavailability in vivo. In some embodiments, the oligonucleotides herein (e.g., ds oligonucleotides) contain an analog of the 5' phosphate that is resistant to such degradation. Examples of such phosphate analogs include, but are not limited to, oxymethylphosphonate, vinylphosphonate, malonylphosphonate, or combinations thereof. In certain embodiments, the 3'-end of the oligonucleotide strand is linked to a chemical moiety ("phosphomimetic") that mimics the electrostatic and steric properties of the natural 5' phosphate group.

[0158] Alternatively or additionally, the oligonucleotides have a phosphate analog at the 4' carbon position of the sugar (referred to as a 4'-phosphate analog). See, for example, International Patent Application Publication No. WO2018 / 045317. In some embodiments, the oligonucleotides herein include a 4'-phosphate analog at the 5' terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate or an analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., to the 4' carbon). In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or aminomethylphosphonate or an analog thereof, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4' carbon of the sugar moiety. In certain embodiments, the 4'-phosphate analog has the formula -O-CH 2 -PO(OH) 2 or O-CH 2 -PO(OR) 2 (Wherein, R is independently H, CH 3 , alkyl group, CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , C.H. 2 OCH 2 CH2 Si(CH 3 ) 3 or a protecting group. In certain embodiments, the alkyl group is selected from 2 CH 3 In other particular embodiments, R is independently H, CH 3 , or C.H. 2 CH 3 is selected from.

[0159] C. Modified Internucleotide Linkages: In addition to the modifications described above, the oligonucleotides herein (e.g., ds oligonucleotides) contain modified internucleotide linkages. In some embodiments, phosphate modifications or substitutions can result in oligonucleotides containing at least one (e.g., at least 1, at least 2, at least 3, or at least 5) modified internucleotide linkages. In some embodiments, the oligonucleotides herein (e.g., ds oligonucleotides) contain from about 1 to about 10 (e.g., 1-10, 2-8, 4-6, 3-10, 5-10, 1-5, 1-3, or 1-2) modified internucleotide linkages. In other embodiments, the oligonucleotides contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

[0160] Examples of modified internucleotide bonds include, but are not limited to, phosphorodithioate bonds, phosphorothioate bonds, phosphotriester bonds, thionoalkylphosphonate bonds, thioalkylphosphotriester bonds, phosphoramidite bonds, phosphonate bonds, and / or boranophosphate bonds.In some embodiments, at least one modified internucleotide bond of any one of the oligonucleotides disclosed herein is a phosphorothioate bond.

[0161] In some embodiments, the oligonucleotides herein include a phosphorothioate bond between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and / or positions 21 and 22 of the antisense strand. In other embodiments, the oligonucleotides include a phosphorothioate bond between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and / or positions 21 and 22 of the antisense strand.

[0162] In certain embodiments, the oligonucleotides herein include: a sense strand having 2'-F modified nucleotides at positions 8-11, 2'-OMe modified nucleotides at positions 1-7, 12-27, and 31-36, GalNAc conjugated nucleotides at positions 28, 29, and 30, and a phosphorothioate linkage between positions 1 and 2; An antisense strand having 2'-F modified nucleotides at positions 2-5, 7, 10, and 14, 2'-OMe at positions 1, 6, 8-9, 11-13, and 15-22, phosphorothioate linkages between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, a 5'-terminal nucleotide containing a 4'-phosphate analog at position 1 (optionally the 5'-terminal nucleotide is 4-O-monomethylphosphonate-2'-O-methyluridine [MePhosph onate-4O-mU); where positions 1-20 of the antisense strand form a duplex region with positions 1-20 of the sense strand, positions 21-36 of the sense strand form a stem-loop, and positions 27-30 form a loop of the stem-loop, optionally, positions 27-30 comprise tetraL, and positions 21 and 22 of the antisense strand comprise overhangs, and wherein the sense and antisense strands are selected from Table 3, optionally from the group consisting of: (a) SEQ ID NOs: 139 and 140; (b) SEQ ID NOs: 147 and 148; (c) SEQ ID NOs: 221 and 222; (d) SEQ ID NOs: 273 and 274; (e) SEQ ID NOs: 321 and 322; (f) SEQ ID NOs: 333 and 334; (g) SEQ ID NOs: 361 and 362.

[0163] In certain other embodiments, the modified sense and antisense strands are selected from Table 4, optionally: (a') SEQ ID NOs: 523 and 524; (b') SEQ ID NOs: 531 and 532; (c') SEQ ID NOs: 605 and 606; (d') SEQ ID NOs: 657 and 658; (e') SEQ ID NOs: 705 and 706; (f') SEQ ID NOs: 717 and 718; (g') Selected from the group consisting of SEQ ID NOs: 745 and 746.

[0164] D. Base Modification: In addition to the above modifications, the oligonucleotides (e.g., ds oligonucleotides) herein also contain one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as base analog) is linked to the 1' position of the nucleotide sugar moiety. In some embodiments, the modified nucleobase is a nitrogenous base. In other embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, US Patent Application Publication No. 2008 / 0274462. In other particular embodiments, the modified nucleobase is a universal base. However, in certain embodiments, the modified nucleotide does not contain a nucleobase (abasic).

[0165] With respect to universal bases, they include heterocyclic moieties located at the 1' position of the nucleotide sugar moiety of a modified nucleotide, or at the equivalent position of a nucleotide sugar moiety substitute, that, when present in a duplex, are positioned opposite more than one type of base without substantially altering the structure of the duplex. Furthermore, compared to a reference ss nucleic acid (e.g., an oligonucleotide) that is fully complementary to a target nucleic acid, a ss nucleic acid having a universal base has a lower T than a duplex formed with a complementary nucleic acid. mHowever, compared to a reference ss nucleic acid in which the universal base is replaced by a base resulting in one mismatch, the ss nucleic acid with the universal base has a higher T than the duplex formed with the nucleic acid with the mismatched base. m It forms a duplex with a target nucleic acid having the following structure:

[0166] Examples of universal binding nucleotides include, but are not limited to, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see, e.g., U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al. (1995) Nucleic Acids Res. 23:4363-70; Loakes et al. (1995) Nucleic Acids Res. 23:2361-66; and Loakes & Brown (1994) Nucleic Acids Res. 22:4039-403).

[0167] E. Reversible Modification: Certain modifications can be made to protect the oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) from the in vivo environment before reaching the target cell, but can also be made to reduce the potency or activity of the oligonucleotide after reaching the cytoplasm of the target cell. Thus, reversible modifications can be made so that the oligonucleotide retains the desired properties outside the cell and is removed when it enters the cytoplasmic environment of the cell. Reversible modifications can be removed, for example, by the action of intracellular enzymes or by intracellular chemical conditions (e.g., by reduction by intracellular glutathione).

[0168] In some embodiments, the reversible modified nucleotide comprises a glutathione-sensitive moiety. Typically, oligonucleotides are chemically modified with cyclic disulfide moieties to mask the negative charge caused by internucleotide diphosphate bonds and improve cellular uptake and nuclease resistance. See US Patent Application Publication No. 2011 / 0294869, International Patent Application Publication No. WO2014 / 088920, and International Patent Application Publication No. WO2015 / 188197, and Meade et al. (2014) Nat. Biotechnol. 32: 1256-63. This reversible modification of internucleotide diphosphate bonds is designed to be cleaved intracellularly by the reductive environment (e.g., glutathione) of the cytosol. An early example was a neutralizing phosphotriester modification, which was reported to be cleavable intracellularly (see, e.g., Dellinger et al. (2003) J. Am. Chem. Soc. 125:940-50).

[0169] Some reversible modifications protect the oligonucleotide during in vivo administration (e.g., transport through blood and / or lysosomal / endosomal compartments of cells) where the oligonucleotide is exposed to nucleases and other harsh environmental conditions (e.g., pH). Upon release into the cytosol of cells, where glutathione levels are higher compared to the extracellular space, the modifications are reversed, resulting in cleaved oligonucleotides. Using reversible glutathione-sensitive moieties, it is possible to introduce sterically larger chemical groups into the oligonucleotide compared to the options available using irreversible chemical modifications. This is because these larger chemical groups are removed in the cytosol and therefore should not interfere with the biological activity of the oligonucleotide in the cytosol of cells. As a result, these larger chemical groups can be engineered to confer various advantages on the oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, reduced immunogenicity, etc. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

[0170] In some embodiments, the glutathione-sensitive moiety is attached to the sugar of the nucleotide. In certain embodiments, the glutathione-sensitive moiety is attached to the 2'-carbon of the sugar of the modified nucleotide. Additionally or alternatively, the glutathione-sensitive moiety is attached to the 5' carbon of the sugar, particularly when the modified nucleotide is the 5' terminal nucleotide of the oligonucleotide. Additionally or alternatively, the glutathione-sensitive moiety is attached to the 3' carbon of the sugar, particularly when the modified nucleotide is the 3' terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group (see, e.g., International Patent Application Publication No. WO2018 / 039364).

[0171] VI. Targeting Ligand: It is desirable to target the oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) to one or more cells or one or more organs. Such a strategy can be useful to avoid undesirable effects in other organs or to avoid unnecessary loss of oligonucleotides to cells, tissues, or organs that do not benefit from the oligonucleotide. Thus, oligonucleotides are modified to facilitate targeting and / or delivery to tissues, cells, or organs (e.g., facilitate delivery of oligonucleotides to the liver). In some embodiments, oligonucleotides are modified to facilitate delivery of oligonucleotides to hepatocytes in the liver. In some embodiments, the oligonucleotides comprise at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) conjugated to one or more targeting ligand(s).

[0172] Targeting ligands include, but are not limited to, carbohydrates, amino sugars, cholesterol, peptides, polypeptides, proteins or protein parts (e.g., antibodies or antibody fragments), or lipids. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand can be an Arg-Gly-Asp (RGD) peptide used to target tumor vasculature or glioma cells, a Cys-Arg-Glu-Lys-Ala (CREKA) peptide for targeting tumor vasculature or tumor stroma, transferrin, lactoferrin, or an aptamer for targeting transferrin receptor expressed on central nervous system (CNS) vasculature, or an anti-EGFR antibody for targeting epidermal growth factor receptor (EGFR) on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

[0173] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of an oligonucleotide may each be conjugated to a separate targeting ligand. In some embodiments, 2-4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In other embodiments, a targeting ligand may be conjugated to 2-4 nucleotides at either the end of the sense strand or the antisense strand (e.g., a targeting ligand is conjugated to a 2-4 nucleotide overhang or extension at the 5' or 3' end of the sense strand or the antisense strand), such that the targeting ligand resembles the bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may include a stem-loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the L of the stem-loop may be individually conjugated to a targeting ligand. In some embodiments, the oligonucleotide comprises a stem-loop at the 3' end of the sense strand, wherein the L of the stem-loop comprises a triL or a tetraL, and each of the three or four nucleotides of the triL or tetraL is individually conjugated to a targeting ligand.

[0174] GalNAc is a high affinity ligand for ASGPR, which is primarily expressed on the sinusoidal surface of hepatocytes, and plays a major role in the binding, internalization, and subsequent excretion of circulating glycoproteins containing terminal galactose or GalNAc residues (asialoglycoproteins). In some embodiments, the oligonucleotides herein can be conjugated (indirectly or directly) to a GalNAc moiety and used to target the oligonucleotide to an ASGPR expressed in a cell. In some embodiments, the oligonucleotide is conjugated to at least one or more GalNAc moieties, and the GalNAc moiety targets the oligonucleotide to an ASGPR expressed on a human liver cell (e.g., a human hepatocyte).

[0175] The oligonucleotide is directly or indirectly conjugated to a monovalent GalNAc. In some embodiments, the oligonucleotide is directly or indirectly conjugated to more than one monovalent GalNAc (i.e., conjugated to two, three, or four monovalent GalNAc moieties, typically three or four monovalent GalNAc moieties). In some embodiments, the oligonucleotide is conjugated to one or more divalent, trivalent, or tetravalent GalNAc moieties.

[0176] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide may each be conjugated to a GalNAc moiety. In some embodiments, 2-4 nucleotides of L are each conjugated to a separate GalNAc. In other embodiments, 1-3 nucleotides of triL are each conjugated to a separate GalNAc. In some embodiments, the targeting ligand is conjugated to 2-4 nucleotides at either end of the sense or antisense strand (e.g., the ligand is conjugated to a 2-4 nucleotide overhang or extension at the 5' or 3' end of the sense or antisense strand), such that the GalNAc moieties resemble toothbrush bristles and the oligonucleotide resembles a toothbrush. In some embodiments, the GalNAc moieties are conjugated to nucleotides in the sense strand. For example, 4 GalNAc moieties are conjugated to nucleotides in L of the sense strand, with each GalNAc moiety being conjugated to one nucleotide. In a particular embodiment, three GalNAc moieties are conjugated to nucleotides within L of the sense strand, and each GalNAc moiety is conjugated to one nucleotide.

[0177] In some embodiments, the oligonucleotides herein comprise GalNAc attached to any one or more nucleotides of a triL or tetraL via any linker described herein, as illustrated below (X=heteroatom): [ka]

[0178] In some embodiments, the oligonucleotides herein contain a monovalent GalNAc attached to a guanine nucleotide, referred to as 2'-aminodiethoxymethanol-guanine-GalNAc or [ademG-GalNAc], as shown below: [ka]

[0179] In some embodiments, the oligonucleotides herein contain a monovalent GalNAc attached to an adenine nucleotide, referred to as 2'-aminodiethoxymethanol-adenine-GalNAc or [ademA-GalNAc], as shown below: [ka]

[0180] An example of such a conjugation is shown below for a tetraL having the nucleotide sequence GAAA in the 5' to 3' direction (L = linker, X = heteroatom). The stem attachment points are indicated. Such tetraLs are, for example, listed in Tables 3 and 4 and are present in positions 27-30 of the sense strand shown in Figure 1. In the formula: [ka] is used to represent the point of attachment to the oligonucleotide chain: [ka]

[0181] The targeting ligand is linked to the nucleotide using an appropriate method or chemical technique (e.g., click chemistry). One method of conjugating the targeting ligand to the nucleotide is to use a click linker. In some embodiments, the targeting ligand is conjugated to any one of the nucleotides of the oligonucleotides herein using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. An example of a loop having the nucleotide GAAA in the 5' to 3' direction, in which a GalNAc moiety is attached to the nucleotide of teraL using an acetal linker, is shown below. Such a loop is present, for example, at positions 27 to 30 of any one of the sense strands listed in Tables 3 or 4. In the formula: [ka] is the point of attachment to the oligonucleotide chain: [ka] [ka]

[0182] In some embodiments, a duplex extension (e.g., up to 3, 4, 5, or 6 bp in length) is placed between the targeting ligand (e.g., the GalNAc moiety) and the oligonucleotide. In other embodiments, the oligonucleotide does not have a GalNAc conjugated to it.

[0183] Formulations and pharmaceutical compositions The oligonucleotides herein (e.g., ds oligonucleotides), or pharma- ceutically acceptable salts thereof (e.g., trifluoroacetate, acetate, or hydrochloride), are incorporated into formulations or pharmaceutical compositions. To facilitate the use of oligonucleotides, various formulations have been developed. For example, oligonucleotides can be delivered to an individual or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or confer other beneficial properties to the oligonucleotides in the formulation. In some embodiments, oligonucleotides are formulated in buffers such as phosphate buffered saline, liposomes, micellar structures, and capsids.

[0184] To enhance in vivo compatibility and effectiveness, oligonucleotides can react with any of a number of inorganic and organic acids / bases to form pharma- ceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and general methods for preparing them are well known in the art (see, for example, Stahl et al., “Handbook of Pharmaceutical Salts: Properties, Selection and Use,” 2 nd Revised Edition (Wiley-VCH, 2011). Pharmaceutically acceptable salts as used herein include sodium, trifluoroacetate, hydrochloride and acetate salts.

[0185] The oligonucleotide formulations herein containing cationic lipids are used to facilitate the transfection of oligonucleotides into cells.For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used.Suitable lipids include oligofectamine (ThermoFisher Technologies), lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene6 (Roche), each of which can be used according to the manufacturer's instructions.

[0186] Thus, in some embodiments, the formulations herein may comprise liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles (such as lipid nanoparticles), or may be otherwise formulated for administration to a cell, tissue, organ, or body of an individual in need thereof (see, e.g., Remington, "The Science and Practice of Pharmacy" (LV Allen Jr., ed., 22 nd Edition, Pharmaceutical Press, 2013).

[0187] In some embodiments, the formulations herein further comprise an excipient, which can provide the composition with improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide), or a vehicle (e.g., buffer, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotides herein are lyophilized to extend shelf life, and then put into solution before use (e.g., administration to an individual). Thus, the excipient in a pharmaceutical composition comprising one or more oligonucleotides is a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone), or a disintegration temperature regulator (e.g., dextran, Ficoll™, or gelatin).

[0188] A pharmaceutical composition is formulated to be compatible with its intended route of administration, including, but not limited to, parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0189] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include, but are not limited to, physiological saline, bacteriostatic water, Cremophor EL™ (BASF) or phosphate buffered saline (PBS). Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, in the composition. Sterile injection solutions are prepared by mixing the required amount of the oligonucleotides herein in a selected solvent, optionally with one or a combination of the above-listed components, followed by filter sterilization.

[0190] Additionally, the pharmaceutical compositions contain at least about 0.1% or more of a therapeutic agent (e.g., one or more oligonucleotides herein), although the percentage of therapeutic agent may be from about 1% to about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0191] Some examples are directed to targeted delivery of at least one of the oligonucleotides described herein to the liver, although targeting to other tissues is also contemplated.

[0192] kit The oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) can be incorporated into a kit that includes one or more of the oligonucleotides herein and instructions for use. In some embodiments, the kit includes one or more oligonucleotides and a package insert that includes instructions for use of the kit and / or any of its components. In other embodiments, the kit includes suitable containers, one or more oligonucleotides, one or more controls, and various buffers, reagents, enzymes, and other standard components known in the art.

[0193] In some embodiments, the container is at least one vial, well, test tube, flask, bottle, syringe, or other container means into which one or more oligonucleotides are placed, and in some embodiments, appropriately dispensed. In other embodiments in which additional components are provided, the kit includes an additional container into which the components are placed. The kit also includes a means for containing one or more oligonucleotides and other reagents in a closed condition suitable for commercial sale. Such containers include injection or blow molded plastic containers into which the desired vials are retained. The container and / or kit includes a label with instructions for use and / or warnings.

[0194] In some embodiments, the kit comprises one or more oligonucleotides herein and a pharma- ceutically acceptable carrier, or a pharmaceutical composition comprising one or more oligonucleotides, and instructions for use in treating or slowing the progression of a disease, disorder, or condition associated with SCAP activity in an individual in need thereof.

[0195] method Method of preparation The oligonucleotides (e.g., ds oligonucleotides, such as RNAi oligonucleotides) herein are prepared using methods and / or techniques known to those skilled in the art, such as, for example, conventional solid-phase nucleic acid synthesis. The polynucleotides of the oligonucleotides are assembled on a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers, including DNA / RNA synthesizers, are commercially available, such as from Applied Biosystems (Foster City, CA), BioAutomation (Irving, TX), and GE Healthcare Life Sciences (Pittsburgh, PA).

[0196] As will be appreciated by those of skill in the art, other methods and / or techniques for synthesizing oligonucleotides may be used. Additionally, the various synthetic steps may be performed in an alternating order or sequence to arrive at the desired compound. Other synthetic chemistry transformations, protecting groups (e.g., hydroxyl, amino, etc. present on bases), and protecting group methodologies (protection and deprotection) useful in the synthesis of oligonucleotides are known in the art and are described, for example, in Larock, "Comprehensive Organic Transformations," VCH Publishers (1989); Greene & Wuts, Protective Groups in Organic Synthesis, 2 nd Ed., John Wiley & Sons (1991); Fieser & Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley & Sons (1994); and Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons (1995).

[0197] How to use I. Methods for reducing SCAP activity in cells, tissues, organs, and organisms: The oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) are used to reduce SCAP mRNA, SCAP protein, and / or SCAP activity in a cell, tissue, organ, or individual. The method includes the steps described herein, which may, but need not, be performed in the order described. However, other orders are contemplated. Furthermore, individual or multiple steps can be performed in parallel and / or overlapping in time, and / or with individual or multiple repeated steps. Additionally, the method includes additional unspecified steps.

[0198] The methods include contacting or delivering an effective amount of any of the oligonucleotides herein to a cell, population of cells, tissue, organ, or individual to reduce SCAP expression. In some embodiments, the reduction in SCAP activity is determined by measuring a reduction in the amount or level of SCAP mRNA, SCAP protein, and / or SCAP activity in the cell.

[0199] For suitable cell types, the cell type is any cell that expresses the mRNA (e.g., hepatocytes, macrophages, monocyte-derived cells, prostate cancer cells, brain, endocrine tissue, bone marrow, lymph nodes, lung, gallbladder, liver, duodenum, small intestine, pancreas, kidney, gastrointestinal tract, bladder, fat and soft tissue, and skin cells). In some embodiments, the cell is a primary cell obtained from an individual. In some embodiments, the primary cell has undergone a limited number of passages, whereby the cell substantially maintains its native phenotypic characteristics. In some embodiments, the cell is ex vivo, in vivo, or in vitro (i.e., one or more oligonucleotides herein can be delivered to the cell in culture or to the organism in which the cell resides).

[0200] In some embodiments, the oligonucleotides herein are delivered to cells or cell populations using nucleic acid delivery methods known in the art, including but not limited to injecting a solution containing the oligonucleotide, bombarding with particles coated with the oligonucleotide, exposing a cell or cell population to a solution containing the oligonucleotide, or electroporating cell membranes in the presence of the oligonucleotide.Other methods known in the art for delivering oligonucleotides to cells can be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection, such as calcium phosphate.

[0201] The reduction in SCAP activity is determined by an assay or technique that evaluates one or more molecules, characteristics, or properties of a cell or population of cells associated with SCAP gene expression (e.g., using a SCAP expression biomarker) or by an assay or technique that evaluates molecules that directly indicate SCAP activity in a cell or population of cells (e.g., SCAP mRNA, SCAP protein, and / or SCAP activity). In some embodiments, the extent to which an oligonucleotide reduces SCAP activity is assessed by comparing SCAP activity in a cell or population of cells contacted with the oligonucleotide to a control cell or population of cells (e.g., a cell or population of cells not contacted with the oligonucleotide or contacted with a control oligonucleotide). In some embodiments, the control amount or level of SCAP activity in a control cell or population of cells is predetermined such that it is not necessary to measure the control amount or level in every instance that an assay or technique is performed. The predetermined level or value can take a variety of forms, including, but not limited to, a single cutoff value, such as a median or mean value.

[0202] Contacting or delivering the oligonucleotides herein to a cell or population of cells reduces SCAP activity. In some embodiments, the reduction in SCAP activity is compared to a control amount or level of SCAP activity in a cell or population of cells not contacted with the oligonucleotide or contacted with a control oligonucleotide. In some embodiments, the reduction in SCAP activity is about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less compared to the control amount or level of SCAP activity. In some embodiments, the control amount or level of SCAP activity is the amount or level of SCAP mRNA, SCAP protein, and / or SCAP activity in a cell or population of cells not contacted with the oligonucleotides herein. In some embodiments, the effect of delivery of oligonucleotides to a cell or population of cells by the methods herein is evaluated after any finite period or period (e.g., minutes, hours, days, weeks, and / or months). For example, SCAP activity is determined in a cell or population of cells for at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, or about 24 hours. Alternatively, SCAP activity is determined in a cell or population of cells for at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or more after contacting or delivering an oligonucleotide to the cell or population of cells. In some embodiments, SCAP activity is determined in a cell or population of cells at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or more after contacting or delivering the oligonucleotide to the cell or population of cells.

[0203] In some embodiments, the oligonucleotides herein are delivered in the form of a transgene engineered to express one or more oligonucleotides or strands (e.g., sense and antisense strands) in a cell. For example, the oligonucleotides are delivered using a transgene designed to express any of the oligonucleotides herein. The transgene may be delivered using a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., a plasmid or synthetic mRNA). In some embodiments, the transgene is directly injected into the individual.

[0204] II. Treatment method Methods of treating an individual having, suspected of having, or at risk of developing a disease, disorder, or condition associated with SCAP activity include administering to the individual at least one or more of the oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides). Furthermore, methods of treating or reducing the onset or progression of a disease, disorder, or condition associated with SCAP activity in an individual include using one or more of the oligonucleotides herein. Furthermore, methods of achieving one or more therapeutic effects in an individual having a disease, disorder, or condition associated with SCAP activity include providing one or more of the oligonucleotides herein. In some embodiments, an individual can be treated by administering a therapeutically effective amount of any one or more of the oligonucleotides herein. In some embodiments, the treatment includes reducing SCAP activity. In some embodiments, the individual is treated therapeutically. In some embodiments, the individual is treated prophylactically. In all of these embodiments, the oligonucleotide of interest is selected from Table 3 or 4.

[0205] In some embodiments, one or more oligonucleotides, or pharmaceutical compositions comprising same, are administered to an individual having a disease, disorder, or condition associated with SCAP activity to reduce SCAP activity in the individual, thereby treating the individual. In some embodiments, the amount or level of SCAP mRNA in the individual is reduced. In other embodiments, the amount or level of SCAP protein in the individual is reduced. In still other embodiments, the amount or level of SCAP activity in the individual is reduced. In still other embodiments, the amount or level of hepatic TG (e.g., one or more TG(s) or total TG in the liver) and / or cholesterol in the individual, particularly in the liver, is reduced. In still other embodiments, the amount or level of hepatic inflammation can be reduced. In still other embodiments, the level of liver fibrosis is reduced. In still other embodiments, the amount or level of plasma aspartate aminotransferase (AST), plasma alanine aminotransferase (ALT), plasma cytokeratin 18 (CK-18), or plasma N-terminal type III collagen propeptide (Pro-C3) is reduced. In any of the above disclosed embodiments, the oligonucleotide comprises a sense strand having the nucleotide sequence of any one of SEQ ID NOs: 532, 531, 605, 657, 705, 717, and 745, and an antisense strand having the nucleotide sequence of any one of SEQ ID NOs: 524, 532, 606, 658, 706, 718, and 746.

[0206] In some embodiments, SCAP activity in the individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to SCAP activity before administration of one or more oligonucleotides or pharmaceutical compositions thereof. In other embodiments, SCAP activity in the individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to SCAP activity in an individual that has not received one or more oligonucleotides or pharmaceutical compositions or that has received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control individual).

[0207] In certain embodiments, the amount or level of SCAP mRNA in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to the amount or level of SCAP mRNA before administration of one or more oligonucleotides or pharmaceutical compositions thereof. In some embodiments, the amount or level of SCAP mRNA in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to the amount or level of SCAP mRNA in an individual (e.g., a reference or control individual) that has not received one or more oligonucleotides or pharmaceutical compositions or that has received a control oligonucleotide, pharmaceutical composition, or treatment.

[0208] In certain embodiments, the amount or level of SCAP protein in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to the amount or level of SCAP protein before administration of one or more oligonucleotides or pharmaceutical compositions thereof. In other embodiments, the amount or level of SCAP protein in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to the amount or level of SCAP protein in an individual that has not received one or more oligonucleotides or pharmaceutical compositions or that has received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control individual).

[0209] In certain embodiments, the amount or level of SCAP activity in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to the amount or level of SCAP activity before administration of one or more oligonucleotides or pharmaceutical compositions thereof. In some embodiments, the amount or level of SCAP activity in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or more than 99% when compared to the amount or level of SCAP activity in an individual that has not received one or more oligonucleotides or pharmaceutical compositions or that has received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control individual).

[0210] In certain embodiments, the amount or level of TG in an individual, particularly hepatic TG, may be reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of TG before administration of one or more oligonucleotides or pharmaceutical compositions thereof. In some embodiments, the amount or level of TG in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of TG in an individual that has not received one or more oligonucleotides or pharmaceutical compositions or that has received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control individual).

[0211] In certain embodiments, the amount or level of cholesterol in an individual, particularly liver cholesterol, may be reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of cholesterol before administration of one or more oligonucleotides or pharmaceutical compositions thereof. In some embodiments, the amount or level of cholesterol in an individual is reduced by at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or greater than 99% when compared to the amount or level of cholesterol in an individual that has not received one or more oligonucleotides or pharmaceutical compositions or that has received a control oligonucleotide, pharmaceutical composition, or treatment (e.g., a reference or control individual).

[0212] Here, the amount or level of SCAP activity, SCAP mRNA, SCAP protein, SCAP activity, hepatic TG, hepatic cholesterol, or any combination thereof is reduced in a cell (e.g., a hepatocyte), a population or group of cells (e.g., an organoid), a tissue (e.g., a liver tissue), a sample (e.g., a liver biopsy sample), an organ (e.g., liver), blood or a fraction thereof (e.g., plasma), or any other biological material obtained or isolated from an individual. In some embodiments, the amount or level of SCAP activity, SCAP mRNA, SCAP protein, SCAP activity, TG, cholesterol, or any combination thereof is reduced in one or more cells (e.g., hepatocytes and one or more other types of cells), one or more groups of cells, one or more tissues (e.g., liver tissue and one or more other types of tissue), one or more samples (e.g., liver biopsy samples and one or more other types of biopsy samples), one or more organs (e.g., liver and one or more other organs), one or more blood fractions (e.g., plasma and one or more other blood fractions) taken or isolated from an individual.

[0213] Examples of diseases, disorders, or conditions associated with SCAP include, but are not limited to, AH, ALD, CCA, cirrhosis, liver fibrosis, hepatitis, HCC, fatty liver, NAFLD, NASH, and PSC, as well as associated diseases, disorders, and conditions in individuals such as, for example, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, ASCVD, diabetes, and / or obesity, or combinations thereof.

[0214] Due to the high specificity of the oligonucleotides herein, the oligonucleotides herein specifically target the mRNA of the target gene in a cell, tissue, or organ (e.g., liver). In the prevention of a disease, the target gene is one that is required for the initiation or maintenance of the disease, or one that has been identified as being associated with a high risk of suffering from the disease. In the treatment of a disease, one or more oligonucleotides herein are contacted with a cell, tissue, or organ that exhibits the disease or is involved in mediating the disease. For example, an oligonucleotide that is substantially complementary to all or a portion of a wild-type (i.e., naturally occurring) or mutant gene associated with a disease, disorder, or condition associated with SCAP activity is contacted with or introduced into a cell or tissue type of interest, such as a hepatocyte or other liver cell.

[0215] In some embodiments, the target gene is from any mammal, such as a human. Any gene can be silenced according to the methods of the present invention. The methods of the present invention typically include administering to an individual a therapeutically effective amount, i.e., an amount that can produce a desired therapeutic result, of one or more of the oligonucleotides of the present invention. A therapeutically acceptable amount is an amount that therapeutically treats a disease or disorder or condition. The appropriate dosage for any one individual depends on certain factors, including the individual's size, body surface area, age, the composition administered, the active ingredient(s) in the composition, the time and route of administration, overall health, and other therapeutic agents administered at the same time.

[0216] In the methods, an individual is administered any one of the oligonucleotides or compositions herein enterally (e.g., orally, by a gastric feeding tube, by a duodenal feeding tube, via a gastrostomy, or rectally), parenterally (e.g., subcutaneous, intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intraventricular, or intrathecal injection), locally (e.g., transdermally, by inhalation, eye drops, or via a mucosa), or by direct injection into a target organ (e.g., the liver of the individual). Typically, the oligonucleotide or composition is administered intravenously or subcutaneously.

[0217] As a non-limiting set of examples, the oligonucleotides or compositions herein are typically administered quarterly (once every three months), bimonthly (once every two months), monthly, or weekly. For example, the oligonucleotides or compositions are administered weekly, or at two-weekly or three-weekly intervals. In certain embodiments, the oligonucleotides or compositions are administered daily. In some embodiments, the individual is administered one or more loading doses of the oligonucleotide or composition, followed by one or more maintenance doses of the oligonucleotide or composition.

[0218] In some embodiments, the individual is a human, NHP, or other mammal, hi other embodiments, the individual is a domestic animal such as a dog or cat, a livestock animal such as a horse, cow, pig, sheep, goat, or chicken, and an animal such as a mouse, rat, guinea pig, or hamster.

[0219] III. Medical Uses: The oligonucleotides herein (e.g., ds oligonucleotides, such as RNAi oligonucleotides) can be used and adapted for use to treat individuals who would benefit from reduced SCAP activity (e.g., humans having a disease, disorder, or condition associated with SCAP activity). In some embodiments, oligonucleotides are provided for use or adapted for use in treating individuals having a disease, disorder, or condition associated with SCAP activity. Oligonucleotides are also provided for use or adapted for use in the manufacture of a medicament or pharmaceutical composition for treating a disease, disorder, or condition associated with SCAP activity. In other embodiments, oligonucleotides are provided or adapted for use to target SCAP mRNA and reduce SCAP activity (e.g., via the RNAi pathway). In other embodiments, oligonucleotides are used or adapted for use to target SCAP mRNA and reduce the amount or level of SCAP mRNA, SCAP protein, and / or SCAP activity (i.e., reduce SCAP activity).

[0220] In some embodiments, the methods include selecting individuals for treatment based on the individual having or being predisposed to a marker (e.g., biomarker) of a disease, disorder, or condition associated with SCAP activity, such as, but not limited to, SCAP mRNA, SCAP protein, SCAP activity, or a combination thereof. Similarly, as described in more detail below, the methods may also include additional steps, such as, for example, measuring or obtaining a baseline value of a marker of SCAP activity (e.g., SCAP protein or other biomarker) and then comparing the obtained value to one or more other baseline values ​​or to a value obtained after administering one or more oligonucleotides to the individual to assess the effectiveness of the treatment. EXAMPLES

[0221] The following non-limiting examples are included for purposes of illustration and not limitation.

[0222] Synthesis of oligonucleotides Example 1: Preparation of ds RNAi oligonucleotides Oligonucleotide synthesis and purification: Example ds RNAi oligonucleotides are chemically synthesized using the methods described herein. Generally, dsRNAi oligonucleotides are synthesized using solid-phase oligonucleotide synthesis methods described for 19-23mer siRNAs (see, e.g., Scaringe et al. (1990) Nucleic Acids Res. 18:5433-41 and Usman et al. (1987) J. Am. Chem. Soc. 109:7845-45; see also U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,008,400; 6,111,086; 6,117,657; 6,353,098; 6,362,323; 6,437,117; and 6,469,158).

[0223] Individual RNA strands were synthesized and HPLC purified according to standard methods (Integrated DNA Technologies). For example, RNA oligonucleotides were synthesized using solid-phase phosphoramidite chemistry, deprotected, and desalted on a NAP-5 column (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques (Damha & Olgivie (1993) Methods Mol. Biol. 20:81-114; Wincott et al. (1995) Nucleic Acids Res. 23:2677-84). Oligomers were purified using ion-exchange high-performance liquid chromatography (IE-HPLC) on an Amersham Source 15Q column (1.0 cm x 25 cm, Amersham Pharmacia Biotech) using a 15-minute linear step gradient. The gradient varied from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris, pH 8.5, and buffer B was 100 mM Tris, pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column, and lyophilized.

[0224] The purity of each oligomer was determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc.). The CE capillary had a 100 μm inner diameter and contained ssDNA 100R Gel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide was injected into the capillary, run at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure as assessed by CE were obtained for use in the experiments described below. The identity of the compounds was verified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager DE™ Biospectometry Workstation (Applied Biosystems) following the manufacturer's recommended protocol. The relative molecular weights of all oligomers were obtained, often within 0.2% of the assumed molecular weight.

[0225] Preparation of duplexes: ssRNA oligomers are resuspended (e.g., at 100 μM concentration) in duplex buffer with 100 mM potassium acetate, 30 mM HEPES (pH 7.5). Complementary sense and antisense strands are mixed in equimolar amounts to obtain a final solution of, e.g., 50 μM duplex. Samples are heated to 100°C for 5 minutes in RNA buffer (IDT) and cooled to room temperature before use. dsRNA oligonucleotides are stored at -20°C. ssRNA oligomers are stored lyophilized or in nuclease-free water at -80°C.

[0226] In vitro function Example 2: Modulation of SCAP expression by RNAi oligonucleotides in vitro - DsiRNA-based compounds Identification of SCAP target sequences: To identify RNAi oligonucleotide inhibitors of SCAP expression, a computer-based algorithm is used to computationally generate SCAP target sequences suitable for assaying inhibition of SCAP expression by the RNAi pathway. The algorithm provides RNAi oligonucleotide antisense strand sequences that are complementary to suitable SCAP target sequences (e.g., SEQ ID NO: 1) in human SCAP mRNA. Some of the antisense strand sequences identified by the algorithm are also complementary to corresponding SCAP target sequences in mouse and NHP SCAP mRNAs (e.g., SEQ ID NOs: 3 and 7, respectively). This generates 192 dsRNAi oligonucleotides (formatted as DsiRNA oligonucleotides), each with a unique antisense strand that has a region of complementarity to the SCAP target sequence identified by the algorithm.

[0227] In vitro cell-based assay: The ability of each of the 192 DsiRNAs to inhibit SCAP expression is determined by an in vitro cell-based assay. Moreover, as shown herein, the nucleotide sequences of the sense and antisense strands of the DsiRNAs have distinct patterns of modified nucleotides and phosphorothioate linkages. Briefly, Huh7 cells stably expressing SCAP are transfected with each DsiRNA (1.0 nM) in separate wells of a multi-well cell culture plate. The cells are maintained for 24 hours after transfection, and then the level of SCAP mRNA remaining from the transfected cells is determined using a TAQMAN®-based qPCR assay. Two qPCR assays, a 3' assay and a 5' assay, are used to determine the mRNA level measured by a HEX probe (e.g., Hs HPRT-517-591 and Hs SFRS9-569-712).

[0228] The results of Huh7 cell-based assays using 192 DsiRNAs are shown in Table 1, where the 192 DsiRNAs have antisense strands that are complementary to human, mouse, and NHP SCAP mRNAs ("triple consensus") or complementary to human and NHP SCAP mRNAs ("double consensus"). Transfections of DsiRNAs that result in 30% or less SCAP mRNA remaining in cells compared to negative controls are considered candidate SCAP expression inhibitors (referred to herein as "hits").

[0229] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0230] Furthermore, primary human hepatocytes, such as 3D spheroids, are transfected with each DsiRNA (100 nM) in separate wells of a multi-well cell culture plate. The cells are maintained for 7 days after transfection, and then the level of SCAP mRNA remaining from the transfected cells is determined using a TAQMAN®-based qPCR assay. Two qPCR assays, a 3' assay and a 5' assay, are used to determine the mRNA level measured by a HEX probe (e.g., Hs HPRT-517-591 and Hs SFRS9-569-712).

[0231] The results of the human hepatocyte 3D spheroid-based assay using DsiRNAs are shown in Table 2. As mentioned above, transfections of DsiRNAs that result in 30% or less of SCAP mRNA remaining in the cells compared to the negative control are considered candidate inhibitors of SCAP expression (referred to herein as "hits").

[0232] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0233] These results demonstrate that DsiRNAs designed to target human SCAP mRNA can inhibit SCAP activity in cells (as determined by reduced amounts of SCAP mRNA in DsiRNA-transfected cells) and that nucleotide sequences containing DsiRNA hits are useful for generating RNAi oligonucleotides that inhibit SCAP activity. Furthermore, these results demonstrate that multiple SCAP target sequences are suitable for RNAi-mediated inhibition of SCAP activity.

[0234] Example 3: Inhibition of SCAP expression by RNAi oligonucleotides in vitro - GalXC™-based compounds

[0235] The DsiRNAs screened in Example 2 are selected for in vitro evaluation as GalXC™-based compounds. Briefly, the nucleotide sequences of the DsiRNAs are used to generate 53 corresponding ds RNAi oligonucleotides containing nicked tetraloop GalNAc conjugate structures (referred to herein as "GalXC™-SCAP oligonucleotides") with a 36-mer sense strand and a 22-mer antisense strand. Furthermore, the nucleotide sequences of the sense and antisense strands of the GalXC™ SCAP oligonucleotides have distinct patterns of modified nucleotides and phosphorothioate linkages (see, for example, FIG. 1 for a schematic diagram of the general structure and chemical modification pattern of the GalXC™ SCAP oligonucleotide). Each of the three adenosine nucleotides of the tetraloop is conjugated to a GalNAc moiety (CAS number: 14131-60-3).

[0236] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

Table 3-10

Table 3-11

Table 3-12

Table 3-13

[0237]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

Table 4-21

Table 4-22

Table 4-23

Table 4-24

Table 4-25

Table 4-26

Table 4-27

Table 4-28

Table 4-29

Table 4-30

[0238] The human stem cell 3D spheroid-based assay described in Example 2 was repeated using the GalXC™-SCAP oligonucleotides in Table 4, and the results are shown in Table 5.

[0239] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0240] In vivo function Example 4: Regulation of SCAP expression by RNAi oligonucleotides in vivo - GalXC™ based compounds Mouse Studies: GalXC™-SCAP oligonucleotides listed in Table 3 (unmodified) and Table 4 (modified) are evaluated in a hydrodynamic injection (HDI) mouse model. In these HDI studies, mice are engineered to transiently express human SCAP mRNA in hepatocytes. A GalXC™-SCAP oligonucleotide control (GalXC™-SCAP oligonucleotide number 42; see SEQ ID NOs: 475 and 476) was used as a benchmark control. Briefly, 6-8 week old female CD-1 mice are treated SQ with GalXC™-SCAP oligonucleotide at a dose of 2 mg / kg. Three days (72 hours) later, mice are HDI'd with a DNA plasmid encoding the full-length human gene under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. Liver samples are harvested one day after plasmid transfer. Total RNA from these mice is subjected to qRT-PCR analysis of SCAP mRNA compared to mice treated with the same volume of PBS alone, and values ​​are normalized for transfection efficiency using the NeoR gene contained in the plasmid.

[0241] As shown in Tables 6-8, many of the GalXC™-SCAP oligonucleotides tested inhibited SCAP activity as judged by a decrease in the amount of SCAP mRNA in liver samples from mice treated with the oligonucleotide compared to mice treated with PBS. The average % of SCAO mRNA remaining in liver samples from mice treated with the GalXC™-SCAP oligonucleotide control is compared to mice treated with PBS. Tables 6-8 show that many of the GalXC™-SCAP oligonucleotides tested inhibited SCAP activity to a greater extent than the GalXC™-SCAP oligonucleotide control.

[0242] [Table 6]

[0243] [Table 7]

[0244] [Table 8]

[0245] The table below shows that the in vivo HDI results are consistent with the in vitro data of Huh7 cells and 3D human spheroids in Example 3.

[0246] Based on the results described above, seven GalXC™-SCAP oligonucleotides were selected for a dose-response study in the HDI mouse model, the results of which are shown in Table 9.

[0247] [Table 9]

[0248] GalXC™-SCAP oligonucleotides exhibit a dose-dependent reduction in human SCAP mRNA expression. GalXC™-SCAP oligonucleotides exhibit an ED of 0.4-0.8 mg / kg for exogenously expressed human SCAP mRNA. 50 has.

[0249] Primate (NHP) Study: Based on the mouse results described above, six GalXC™-SCAP oligonucleotides were selected for evaluation of their ability to inhibit SCAP activity in a single-dose (2 or 6 mg / kg), 84-day study in NHPs (e.g., rhesus macaques; Macaca mulatta). Here, NHPs are grouped so that the mean body weight (approximately 5.4 kg) is comparable between control and experimental groups. Each cohort includes six individuals (three males and three females). GalXC™-SCAP oligonucleotides are administered SQ on study day 0. Blood samples are taken at two time points pre-dose (i.e., day -21 and day 0), then weekly post-dose for liver enzyme panel and lipid profile. Ultrasound-guided core needle liver biopsies are taken on study days -21, 28, 56, and 83. At each time point, total RNA from liver biopsy samples was analyzed by qRT-PCR separately to measure SCAP mRNA in monkeys treated with oligonucleotides compared to monkeys treated with an equivalent amount of PBS. To normalize the data, measurements are performed against the geometric mean of two reference genes, PPIB and 18S rRNA. As shown in Table 10, treatment of NHPs with GalXC™-SCAP oligonucleotides inhibits SCAP activity in the liver, as determined by the reduced amount of SCAP mRNA in liver samples from oligonucleotide-treated NHPs compared to PBS-treated NHPs. At all time points evaluated, GalXC™-SCAP oligonucleotides inhibit SCAP activity to a greater extent than benchmark PBS and time-matched controls.

[0250] [Table 10]

[0251] [Table 11]

[0252] [Table 12]

[0253] Approximately 70% reduction in SCAP mRNA is achieved following a single 6 mg / kg administration of GalXC™-SCAP oligonucleotides nos. 70 and 163, approximately 60% reduction in SCAP mRNA is achieved following a single 6 mg / kg administration of GalXC™-SCAP oligonucleotides nos. 107, 157, and 177, and approximately 50% reduction in SCAP mRNA is achieved following a single 6 mg / kg administration of GalXC™-SCAP oligonucleotide no. 66. Additionally, a dose response is observed following administration of 2 mg / kg and 6 mg / kg of GalXC™-SCAP oligonucleotide no. 107, where the ED 50 is approximately 2 mg / kg. Similarly, a sustained reduction in hepatic SCAP mRNA expression is observed 56 days after a single 6.0 mg / kg dose of GalXC™-SCAP oligonucleotide. Approximately 50% reduction in SCAP mRNA is observed 84 days after a single dose of GalXC™-SCAP oligonucleotides nos. 70 and 163.

[0254] Taken together, these results demonstrate that GalXC™-SCAP oligonucleotides designed to target human and NHP SCAP mRNA inhibit SCAP activity in vivo (as determined by a reduction in the amount of SCAP mRNA in treated animals).

[0255] array The following nucleic acid and / or amino acid sequences are referred to in this disclosure and are provided below for reference:

[0256] SEQ ID NO:1 - Wild type human SCAP (4254 bp; NCBI reference sequence NM_012235.4)

[0257] SEQ ID NO:2 - Wild type human SCAP (1279aa; NCBI reference sequence NP_036367.2)

[0258] SEQ ID NO:3 - Wild type mouse SCAP (4226 bp; NCBI reference sequence NM_001001144.3)

[0259] SEQ ID NO:4 - Wild type mouse SCAP (1276 aa; NCBI reference sequence NP_001001144.2)

[0260] SEQ ID NO:5 - Wild type rat SCAP (4281 bp; NCBI reference sequence NM_001100966.2)

[0261] SEQ ID NO:6 - Wild type rat SCAP (1276 aa; NCBI reference sequence NP_001094436.1)

[0262] SEQ ID NO:7 - Wild type non-human primate SCAP (4135 bp; NCBI reference sequence XM_001100342)

[0263] SEQ ID NO:8 - Wild type non-human primate SCAP (1229aa; NCBI reference sequence XP_001100342.2)

[0264]

Table 13-1

Table 13-2

Table 13-3

Table 13-4

Table 13-5

Table 13-6

Table 13-7

Table 13-8

Table 13-9

Table 13-10

Table 13-11

Table 13-12

Table 13-13

[0265]

Table 14-1

Table 14-2

Table 14-3

Table 14-4

Table 14-5

Table 14-6

Table 14-7

Table 14-8

Table 14-9

Table 14-10

Table 14-11

Table 14-12

Table 14-13

Table 14-14

Table 14-15

Table 14-16

Table 14-17

Table 14-18

Table 14-19

Table 14-20

Table 14-21

Table 14-22

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Table 14-24

Table 14-25

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Table 14-31

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Table 14-33

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Table 14-35

Table 14-36

[0266] SEQ ID NO:777 - Target sequence 1 ACATCATCTTGTTTGCCTA

[0267] SEQ ID NO:778 - Target sequence 2 TCTTGTTTGCCTACATCTA

[0268] SEQ ID NO:779 - Target sequence 3 CTTCAGATGCTTTTTTCA

[0269] SEQ ID NO:780 - Target sequence 4 CGCTCTCAGCTATTACAA

[0270] SEQ ID NO:781 - Target sequence 5 CTTAATTGACACCAACTTT

[0271] SEQ ID NO:782 - Target sequence 6 TCAACGGTTCCCTTGATTT

[0272] SEQ ID NO:783 - Target sequence 7 CATCAAGTTCTACTCCATT

[0273] SEQ ID NO: 784 - Artificial sequence GCAGCCGAAAGGCUGC

[0274] [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5]

Table 15-6

Table 15-7

Table 15-8

Table 15-9

Claims

1. RNAi oligonucleotides for regulating the activity of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), wherein the oligonucleotide comprises a sense strand and an antisense strand, and the sense strand and antisense strand form a double helix. The sense strand is 5'-[mUs][mC][mA][mA][mC][mG][mG][fU][fU][fC][fC][mC][mU][ mU][mG][mA][mU][mU][mU][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalN Ac][ademA-GalN This is Ac][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 717), The antisense chain is 5'-[MePhosphonate-4O-mUs][fAs][fAs][fA][fU][mC][fA][mA][mG][fG][mG][mA][mA][fC][mC][mG][mU][mU][mG][mAs][mGs][mG]-3' (SEQ ID NO: 718), mA represents 2'-OMe adenosine; mC represents 2'-OMe cytosine; mG represents 2'-OMe guanosine; mU represents 2'-OMe uridine; fA represents 2'-F adenosine; fC represents 2'-F cytosine; fG represents 2'-F guanosine; fU represents 2'-F-uridine; mAs represents the 2'-OMe adenosine and 3'-phosphorothioate bond; mGs represents the 2'-OMe guanosine and 3'-phosphorothioate bond; mUs represents the 2'-OMe uridine and 3'-phosphorothioate bond; fAs represents a 2'-F adenosine and 3'-phosphorothioate bond; ademA-GalNAc represents 2'-aminodiethoxymethanol-adenine-GalNAc; and MePhosphonate-4O-mUs is, 【Chemistry 1】 The RNAi oligonucleotide that represents the above.

2. A pharmaceutical composition comprising an RNAi oligonucleotide, wherein the oligonucleotide comprises a sense strand and an antisense strand, and the sense strand and antisense strand form a double helix. The sense strand is 5'-[mUs][mC][mA][mA][mC][mG][mG][fU][fU][fC][fC][mC][mU][ mU][mG][mA][mU][mU][mU][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalN Ac][ademA-GalN This is Ac][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 717), The antisense chain is 5'-[MePhosphonate-4O-mUs][fAs][fAs][fA][fU][mC][fA][mA][mG][fG][mG][mA][mA][fC][mC][mG][mU][mU][mG][mAs][mGs][mG]-3' (SEQ ID NO: 718), mA represents 2'-OMe adenosine; mC represents 2'-OMe cytosine; mG represents 2'-OMe guanosine; mU represents 2'-OMe uridine; fA represents 2'-F adenosine; fC represents 2'-F cytosine; fG represents 2'-F guanosine; fU represents 2'-F-uridine; mAs represents the 2'-OMe adenosine and 3'-phosphorothioate bond; mGs represents the 2'-OMe guanosine and 3'-phosphorothioate bond; mUs represents the 2'-OMe uridine and 3'-phosphorothioate bond; fAs represents a 2'-F adenosine and 3'-phosphorothioate bond; ademA-GalNAc represents 2'-aminodiethoxymethanol-adenine-GalNAc; and MePhosphonate-4O-mUs is, 【Chemistry 2】 The pharmaceutical composition that represents the above.

3. The pharmaceutical composition according to claim 2, further comprising a carrier suitable for intravenous administration.

4. The pharmaceutical composition according to claim 3, wherein the carrier comprises water.

5. The pharmaceutical composition according to claim 3, wherein the carrier comprises phosphate-buffered saline.

6. An oligonucleotide according to claim 1 or a pharmaceutical composition according to any one of claims 2 to 5, for use in reducing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) activity in cells, a population of cells, or an object.

7. The oligonucleotide or pharmaceutical composition according to claim 6, wherein the subject has a disease, disorder, or condition related to SCAP activity.

8. The oligonucleotide or pharmaceutical composition according to claim 7, wherein the disease, disorder, or condition associated with SCAP activity is selected from the group consisting of acute coronary artery disease (ACD), atherosclerotic cardiovascular disease (ASCVD), alcoholic hepatitis (AH), alcoholic liver disease (ALD), cardiovascular metabolic disease, cholangiocarcinoma (CCA), cirrhosis, coronary heart disease (CHD), diabetes mellitus, hepatic fibrosis, hepatitis, hepatocellular carcinoma (HCC), hyperlipidemia, hypertriglyceridemia, high non-HDL cholesterol, insulin resistance, hepatic steatosis, metabolic syndrome (MetS), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), obesity, and primary sclerosing cholangitis (PSC).

9. The oligonucleotide or pharmaceutical composition according to claim 6, wherein the reduction of SCAP activity includes reducing the amount or level of SCAP mRNA, the amount or level of SCAP protein, or both.

10. The oligonucleotide or pharmaceutical composition according to claim 9, wherein the subject has a disease, disorder, or condition related to SCAP activity.

11. The oligonucleotide or pharmaceutical composition according to claim 10, wherein the disease, disorder, or condition associated with SCAP activity is selected from the group consisting of acute coronary artery disease (ACD), atherosclerotic cardiovascular disease (ASCVD), alcoholic hepatitis (AH), alcoholic liver disease (ALD), cardiovascular metabolic disease, cholangiocarcinoma (CCA), cirrhosis, coronary heart disease (CHD), diabetes mellitus, hepatic fibrosis, hepatitis, hepatocellular carcinoma (HCC), hyperlipidemia, hypertriglyceridemia, high non-HDL cholesterol, insulin resistance, hepatic steatosis, metabolic syndrome (MetS), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), obesity, and primary sclerosing cholangitis (PSC).

12. The oligonucleotide or pharmaceutical composition according to claim 6, characterized in that the oligonucleotide or pharmaceutical composition is administered to the subject in combination with a second composition or therapeutic agent.

13. An in vitro method for reducing SCAP activity in cells or a population of cells, comprising the step of contacting the cells or population of cells with an oligonucleotide according to claim 1 or a pharmaceutical composition according to any one of claims 2 to 5.

14. The method according to claim 13, wherein the reduction of SCAP activity includes reducing the amount or level of SCAP mRNA, the amount or level of SCAP protein, or both.

15. A kit comprising an oligonucleotide according to claim 1 or a pharmaceutically acceptable salt thereof, any pharmaceutically acceptable carrier, and a package insert containing instructions for administration to subjects having a disease, disorder, or condition related to sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) activity.

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