Rnai constructs for inhibiting SCAP expression and methods of use thereof

Abstract

SCAP (SREBP (sterol response element binding protein) To provide a new approach for reducing the level of a truncated and activated protein and treating hepatological diseases such as nonalcoholic fatty liver disease.SOLUTION: The present invention relates to RNAi constructs for reducing the expression of the SCAP gene. Also described are methods of treating or preventing liver disease, non-alcoholic fatty liver disease (NAFLD), using such RNAi constructs.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for modulating hepatic expression of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), particularly to nucleic acid-based therapeutics for reducing SCAP expression via RNA interference (RNAi), and methods of using such nucleic acid-based therapeutics to treat or prevent liver disease, such as non-alcoholic fatty liver disease (NAFLD).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 854,433, filed May 30, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, encompassing a variety of liver pathologies. Its prevalence has doubled over the past 20 years, and it is now estimated to affect approximately 20% of the global population (Sattar et al. (2014) BMJ 349:g4596; Loomba and Sanyal (2013) Nature Reviews Gastroenterology & Hepatology 10(11):686-690; Kim and Kim (2017) Clin Gastroenterol Hepatol 15(4):474-485; Petta et al. (2016) Dig Liver Dis 48(3):333-342). NAFLD begins with the accumulation of triglycerides in the liver and is defined by the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes in individuals who 1) have no history of significant alcohol consumption and 2) have excluded other types of liver disease (Zhu et al. (2016) World J Gastroenterol 22(36):8226-33; Rinella (2015) JAMA 313(22):2263-73; Yki-Jarvinen (2016) Diabetologia 59(6):1104-11). In some individuals, the accumulation of ectopic fat in the liver, called steatosis, triggers inflammation and hepatocellular injury, leading to a more advanced stage of the disease called nonalcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, it is estimated that 75 to 100 million Americans have NAFLD; NASH accounts for approximately 10 to 30% of NAFLD diagnoses (Rinella, op. cit.; Younossi et al (2016) Hepatology 64(5):1577-1586).

[0004] SCAP (SREBP cleavage-activating protein) is the only known post-transcriptional regulator of the SREBP family of transcription factors. The SREBP (sterol response element-binding protein) family plays an important role in regulating de novo lipogenesis and TG accumulation in the liver. SREBP is synthesized as an inactive precursor in the ER. Immediately after synthesis, SCAP forms a complex with SREBP and escorts SREBP to Golgi vesicles. SREBP is then further processed to release the active amino terminus of the transcription factor. Active SREBP translocates to the nucleus, binds to SREBP response elements, and drives transcriptional activation of target genes (Brown, MS, and Goldstein, JL (1997) Cell 89, 331-340). Targeted silencing of SCAP has been proposed to prevent active SREBP processing and downstream transcriptional changes.

[0005] The SREBP family of proteins contains three isoforms with distinct but overlapping functions: SREBP-1a, SREBP-1c, and SREBP-2. SREBP-1c is abundant in the liver and primarily activates fatty acid and TG synthesis. Germline deficiency of SREBP-1 shows a concomitant increase in SREBP-2 levels, which compensates for the loss of SREBP-1.

[0006] SREBP-2 drives cholesterol production and LDL processing by activating the LDL receptor (LDLR). SREBP-2 also regulates the secreted protein PCSK9, which interacts with LDLR and promotes its degradation, thereby reducing cholesterol absorption. Loss of SCAP / SREBP maintains LDLR protein levels. SREBP1c is the only known transcriptional regulator of PNPLA3. The PNPLA3 polymorphism rs738409 (I148M) is a major genetic determinant for NASH / NAFLD, present in 50% of patients. Silencing SCAP activity has been proposed to benefit individuals with this mutation. Therefore, novel therapeutic agents targeting SCAP function offer a novel approach to reduce SCAP levels and treat hepatological diseases such as nonalcoholic fatty liver disease. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sattar et al. (2014)BMJ 349:g4596 [Non-patent document 2] Loomba and Sanyal(2013)Nature Reviews Gastroenterology & hepatology 10(11):686-690 [Non-patent document 3] Kim and Kim(2017)Clin Gastroenterol Hepatol 15(4):474-485 [Non-patent document 4] Petta et al. (2016) Dig Liver Dis 48(3):333-342) [Non-patent document 5] Zhu et al (2016) World J Gastroenterol 22(36):8226-33 [Non-patent document 6] Rinella (2015) JAMA 313(22):2263-73 [Non-Patent Document 7] Yki-Jarvinen (2016) Diabetologia 59(6):1104-11 [Non-patent document 8] Younossi et al (2016) Hepatology 64(5):1577-1586 [Non-Patent Document 9] Brown, MS, and Goldstein, JL (1997) Cell 89, 331-340. Summary of the Invention [Means for solving the problem]

[0008] The present invention is based, in part, on the design and generation of RNAi constructs that target the SCAP gene and reduce SCAP expression in hepatocytes. Sequence-specific inhibition of SCAP expression is useful for treating or preventing conditions associated with SCAP expression, such as liver-related diseases such as simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis (irreversible, advanced scarring of the liver), or SCAP-associated obesity. Thus, in one embodiment, the present invention provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence complementary to the SCAP mRNA sequence. In a specific embodiment, the antisense strand comprises a region having at least 15 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2.

[0009] In some embodiments, the sense strand of an RNAi construct described herein comprises a sequence sufficiently complementary to that of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, the sense and antisense strands are each about 15 to 30 nucleotides in length. In some embodiments, the RNAi construct comprises at least one blunt end. In other embodiments, the RNAi construct comprises at least one nucleotide overhang. Such a nucleotide overhang may comprise at least 1 to 6 unpaired nucleotides and may be located at the 3'-end of the sense strand, the 3'-end of the antisense strand, or the 3'-ends of both the sense and antisense strands. In certain embodiments, the RNAi construct comprises two unpaired nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand. In other embodiments, the RNAi construct comprises two unpaired nucleotide overhangs at the 3'-end of the antisense strand and blunt ends at the 3'-end of the sense strand / 5'-end of the antisense strand.

[0010] The RNAi constructs of the present invention may contain one or more modified nucleotides, including nucleotides with modifications to the ribose ring, nucleobase, or phosphodiester backbone. In some embodiments, the RNAi constructs contain one or more 2'-modified nucleotides. Such 2'-modified nucleotides may include 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), glycol nucleic acids (GNAs), inverted bases (e.g., inverted adenosines), or combinations thereof. In a specific embodiment, the RNAi constructs contain one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof. In some embodiments, all nucleotides in the sense and antisense strands of the RNAi construct are modified nucleotides.

[0011] In some embodiments, the RNAi construct comprises at least one backbone modification, such as a modified internucleotide or internucleoside bond. In certain embodiments, the RNAi construct described herein comprises at least one phosphorothioate internucleotide bond. In certain embodiments, the phosphorothioate internucleotide bond can be located at the 3'-end or 5'-end of the sense strand and / or antisense strand.

[0012] In some embodiments, the antisense and / or sense strands of an RNAi construct of the invention can comprise or consist of sequences derived from the antisense and sense sequences listed in Table 1 or Table 2. In certain embodiments, the RNAi construct can be any one of the double-stranded compounds listed in any one of Tables 1-2. [Brief explanation of the drawings]

[0013] [Figure 1] Figures 1A-1F show the in vivo effects of SCAP siRNA molecules in mice using the amylin (AMLN) model; (A) shows hepatic SCAP mRNA expression; (B) shows end-stage liver weight / body weight ratio; (C) shows hepatic triglycerides; (D) shows serum PCSK9 levels; (E) shows liver fibrosis pathology readout; and (F) shows aSMA staining as a marker of hepatic stellate cell activation. [Figure 2] Figures 2A-2F show the in vivo effects of SCAP siRNA molecules in mice using the ALIOS model; (A) shows hepatic SCAP mRNA expression; (B) shows end-stage liver weight / body weight ratio; (C) shows hepatic triglycerides; (D) shows serum PCSK9 levels; (E) shows liver fibrosis pathology readout; and (F) shows aSMA staining as a marker of hepatic stellate cell activation. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to compositions and methods for regulating expression of the SREBP cleavage-activating protein (SCAP) gene. In some embodiments, the gene can be in a subject, such as a cell or a mammal (e.g., a human). In some embodiments, the compositions of the present invention comprise an RNAi construct that targets SCAP mRNA and reduces SCAP expression in a cell or mammal. Such RNAi constructs are useful for treating or preventing various forms of liver-related disease, such as simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis (irreversible, advanced scarring of the liver), or SCAP-associated obesity.

[0015] NASH / NAFLD patient populations show increased expression and transcriptional activity of SREBP1c and its target genes (Higuchi et al. (2008) Hepatol Res 38, 1122-1129). Studies using mouse genetics and siRNA-mediated silencing have shown that liver-specific ablation of SCAP activity dramatically reduces hepatic triglyceride content in wild-type and Ob / Ob mice and hamsters fed a high-fat diet. This is accompanied by reduced VLDL secretion and reduced plasma triglyceride levels after SCAP silencing. However, body weight, insulin, and glucose levels remain unchanged (Moon et al. (2012) Cell Metab 15, 240-246). More recent published findings have shown that siRNA silencing of SCAP in mice and dyslipidemic rhesus monkeys significantly reduces TG levels (Jensen et al. (2016) J Lipid Res 57, 2150-2162; Murphy et al. (2017) Metabolism 71, 202-212). Based on these published reports, the inventors hypothesized that administration of siSCAP in NASH patients would alleviate hepatic steatosis and prevent further progression of fibrosis.

[0016] RNA interference (RNAi) is a process in which foreign RNA is introduced into cells to cause the specific degradation of mRNA encoding a targeted protein, thereby reducing protein expression. Advances in both RNAi technology and liver delivery, as well as the increasing success of other RNAi-based therapies, suggest RNAi as a compelling means of therapeutically treating NAFLD by directly targeting SCAP. The inhibitory effects of these sequences were confirmed by screening in Hep3B cells. Using C57B16 mice, the inventors then demonstrated that treatment with SCAP siRNA reduced SCAP expression in mice.

[0017] The term "RNAi construct" as used herein refers to an agent comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of a target gene (e.g., SCAP) via the RNA interference mechanism. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., a messenger RNA or mRNA molecule) in a sequence-specific manner, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other to hybridize and form a duplex region. "Hybridizing" or "hybridization" refers to the pairing of complementary polynucleotides, typically via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. The strand containing a region having a sequence substantially complementary to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand." "Sense strand" refers to the strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand can include a region that has substantial sequence identity to a target sequence.

[0018] In some embodiments, the present invention provides RNAi constructs directed to SCAP. In some embodiments, the present invention includes RNAi constructs containing any of the sequences found in Table 1 or Table 2.

[0019] Double-stranded RNA molecules may contain chemical modifications to ribonucleotides, including modifications to the ribose sugar, base, or backbone components of ribonucleotides, such as those described herein or known in the art. All modifications used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term "double-stranded RNA" for purposes of this disclosure.

[0020] As used herein, a first sequence is "complementary" to a second sequence if, under certain conditions, such as physiological conditions, a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a duplex region. Other such conditions may include moderate or stringent hybridization conditions known to those of skill in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the polynucleotide comprising the first sequence base pairs with the polynucleotide comprising the second sequence without any mismatches along the entire length of one or both nucleotide sequences. A sequence is "substantially complementary" to a target sequence if the sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target sequence. The percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to the bases at corresponding positions in a second sequence or target sequence by the total length of the first sequence. A sequence can also be said to be substantially complementary to another sequence if there are 5, 4, 3, 2, or 1 mismatches or less across a 30-base pair duplex region when the two sequences hybridize. Generally, regardless of the number of nucleotide overhangs as defined herein, the sequence of such overhangs is not taken into account when determining the degree of complementarity between two sequences. For example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand that hybridize to form a 19-base pair duplex region with a 2-nucleotide overhang at the 3' end of each strand would be considered fully complementary as the term is used herein.

[0021] In some embodiments, the region of the antisense strand comprises a sequence that is completely complementary to a region of the target RNA sequence (e.g., SCAP mRNA). In such embodiments, the sense strand may comprise a sequence that is completely complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, e.g., a sequence that has 1, 2, 3, 4, or 5 mismatches within the duplex region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g., within 6, 5, 4, 3, 2, or 1 nucleotide of the 5' and / or 3' ends of the strands). In one embodiment, any mismatches within the duplex region formed by the sense strand and the antisense strand occur within 6, 5, 4, 3, 2, or 1 nucleotide of the 5' end of the antisense strand.

[0022] In certain embodiments, the sense strand and antisense strand of the double-stranded RNA hybridize to form a duplex region, but can otherwise be two separate unconnected molecules. Such double-stranded RNA molecules formed from two separate strands are called "small interfering RNA" or "short interfering RNA" (siRNA). Thus, in some embodiments, the RNAi construct of the present invention comprises siRNA.

[0023] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules do not need to be covalently linked, but can be.When the two strands are covalently linked between the 3'-end of one strand and the 5'-end of the other strand that form a duplex structure by means other than an uninterrupted chain of nucleotides, this bond structure is called "linker".The RNA strands can have the same number of nucleotides or different numbers of nucleotides.The maximum number of base pairs in a duplex is the number of nucleotides of the shortest strand of dsRNA minus any overhangs present in the duplex.In addition to the duplex structure, RNAi can also include one or more nucleotide overhangs.

[0024] In other embodiments, the sense and antisense strands that hybridize to form the duplex region may be part of a single RNA molecule; i.e., the sense and antisense strands are part of the self-complementary region of the single RNA molecule. In such cases, the single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by a flanking sequence of unpaired nucleotides, forming the loop region. The loop region is typically long enough to allow the RNA molecule to fold back on itself so that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region may contain about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Such RNA molecules with at least a partially self-complementary region are referred to as "short hairpin RNAs" (shRNAs). In some embodiments, the loop region may contain at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In some embodiments, the loop region may have 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer unpaired nucleotides. In certain embodiments, the RNAi construct of the present invention comprises an shRNA. The length of the single, at least partially self-complementary RNA molecule may be about 35 to about 100 nucleotides, about 45 to about 85 nucleotides, or about 50 to about 60 nucleotides, and may include a duplex region and a loop region, each having a length as recited herein.

[0025] In some embodiments, the RNAi constructs of the present invention comprise a sense strand and an antisense strand, and the antisense strand comprises a region having a sequence substantially or completely complementary to the SCAP messenger RNA (mRNA) sequence. As used herein, "SCAP mRNA sequence" refers to any messenger RNA sequence, including splice variants, encoding a SCAP protein, including variants or isoforms of SCAP protein from any species (e.g., mouse, rat, non-human primate, human).

[0026] The SCAP mRNA sequence also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. A cDNA sequence refers to the sequence of an mRNA transcript expressed as DNA bases (e.g., guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil, and cytosine). Thus, the antisense strand of an RNAi construct of the present invention can include a region having a sequence substantially or completely complementary to the target SCAP mRNA sequence or SCAP cDNA sequence. The SCAP mRNA or cDNA sequence can include, but is not limited to, any SCAP mRNA or cDNA sequence that can be derived from the NCBI reference sequence for human SCAP (NM_012235) or the NCBI reference sequence for mouse SCAP (NM_001001144).

[0027] The region of the antisense strand can be substantially or fully complementary to at least 15 contiguous nucleotides of the SCAP mRNA sequence. In some embodiments, the target region of the SCAP mRNA sequence to which the antisense strand includes a region of complementarity may span about 15 to about 30 contiguous nucleotides, about 16 to about 28 contiguous nucleotides, about 18 to about 26 contiguous nucleotides, about 17 to about 24 contiguous nucleotides, about 19 to about 25 contiguous nucleotides, about 19 to about 23 contiguous nucleotides, or about 19 to about 21 contiguous nucleotides. In certain embodiments, the region of the antisense strand that includes a sequence substantially or fully complementary to the SCAP mRNA sequence may, in some embodiments, include at least 15 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In other embodiments, the antisense sequence includes at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In some embodiments, the sense and / or antisense sequences comprise at least 15 nucleotides with no more than 1, 2, or 3 nucleotide mismatches from a sequence listed in Table 1 or Table 2.

[0028] The sense strand of an RNAi construct typically contains a sequence sufficiently complementary to that of the antisense strand such that the two strands hybridize under physiological conditions to form a duplex region. A "duplex region" refers to regions within two complementary or substantially complementary polynucleotides that base pair with each other through either Watson-Crick base pairing or other hydrogen-bonding interactions to generate a duplex between the two polynucleotides. The duplex region of an RNAi construct should be of sufficient length to allow the RNAi construct to enter the RNA interference pathway, for example, by engaging the Dicer enzyme and / or the RISC complex. For example, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths of the duplex region within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the duplex region is about 17 to about 24 base pairs in length. In another embodiment, the duplex region is about 19 to about 21 base pairs in length.

[0029] In some embodiments, the RNAi constructs of the present invention contain a duplex region of about 24 to about 30 nucleotides that interacts with a target RNA sequence, e.g., a SCAP target mRNA sequence, leading to cleavage of the target RNA. Without being bound by theory, long double-stranded RNA introduced into cells can be degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Then, siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107: 309). Once it binds to the appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15: 188).

[0030] For embodiments in which the sense and antisense strands are two separate molecules (e.g., the RNAi construct comprises an siRNA), the sense and antisense strands need not be the same length as the duplex region. For example, one or both strands may be longer than the duplex region and may have one or more unpaired nucleotides or mismatches flanking the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to an unpaired nucleotide or a nucleotide that extends beyond the duplex region at the end of a strand. Nucleotide overhangs are typically generated when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of a nucleotide overhang is generally 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, or 2 to 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some embodiments, the overhang comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the overhang comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide.

[0031] The nucleotide overhangs may be at the 5'-end or 3'-end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the sense strand. In some embodiments, the RNAi construct comprises nucleotide overhangs at the 5'-end of the sense strand and the 5'-end of the antisense strand. In other embodiments, the RNAi construct comprises nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand.

[0032] An RNAi construct may comprise a single nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense and antisense strands are perfectly base-paired at the ends of the molecule, with no unpaired nucleotides extending beyond the duplex region. In some embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, an RNAi construct comprises blunt ends at both ends of the double-stranded RNA molecule. In such embodiments, the sense and antisense strands have the same length, and the duplex region is the same length as the sense and antisense strands (i.e., the molecule is duplex throughout its entire length).

[0033] The sense strand and the antisense strand may each independently be about 15 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and the antisense strand are each about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and the antisense strand have the same length but form a duplex region that is shorter than the other strands, such that the RNAi construct has a two-nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand, each 21 nucleotides in length, (ii) a duplex region that is 19 base pairs in length, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand, each 23 nucleotides in length, (ii) a duplex region that is 21 base pairs in length, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the sense strand and the antisense strand have the same length and form a duplex region over the entire length, so that there are no nucleotide overhangs at either end of the double-stranded molecule. In one such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand, each 21 nucleotides in length, and (ii) a duplex region that is 21 base pairs in length. In another such embodiment, the RNAi construct is blunt-ended and comprises (i) sense and antisense strands that are each 23 nucleotides in length, and (ii) a duplex region that is 23 base pairs in length.

[0034] In other embodiments, the sense strand or antisense strand is longer than the other strand, and the two strands form a duplex region having a length equal to the length of the shorter strand, such that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand that is 19 nucleotides long, (ii) an antisense strand that is 21 nucleotides long, (iii) a duplex region that is 19 base pairs long, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand that is 21 nucleotides long, (ii) an antisense strand that is 23 nucleotides long, (iii) a duplex region that is 21 base pairs long, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3'-end of the antisense strand.

[0035] The antisense strand of an RNAi construct of the present invention may comprise any one of the antisense sequences listed in Table 1 or Table 2, or the sequence of nucleotides 1-19 or 1-21 of any of these antisense sequences. Each of the antisense sequences listed in Table 1 and Table 2 comprises a sequence of 19 contiguous nucleotides (the first 19 nucleotides counting from the 5' end) that is complementary to the SCAP mRNA sequence, in addition to a two-nucleotide overhang sequence. Thus, in some embodiments, the antisense strand comprises the sequence of nucleotides 1-19 of any one of the even-numbered sequences of SEQ ID NOS: 2-160, 162-320, 322-462, or 464-604. In some embodiments, the sense strand comprises the sequence of nucleotides 1-19 of any one of the odd-numbered sequences of SEQ ID NOS: 1-159, 161-319, 321-461, or 463-603. In a specific embodiment, the antisense sequence has SEQ ID NO: 82. In certain embodiments, the antisense sequence has SEQ ID NO: 242. In certain embodiments, the antisense sequence has SEQ ID NO: 84. In certain embodiments, the antisense sequence has SEQ ID NO: 244. In certain embodiments, the antisense sequence has SEQ ID NO: 86. In certain embodiments, the antisense sequence has SEQ ID NO: 246. In certain embodiments, the antisense sequence has SEQ ID NO: 88. In certain embodiments, the antisense sequence has SEQ ID NO: 248. In certain embodiments, the antisense sequence has SEQ ID NO: 90. In certain embodiments, the antisense sequence has SEQ ID NO: 250.

[0036] Modified Nucleotides The RNAi construct of the present invention may contain one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide having one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, nor deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, the RNAi construct may contain a combination of modified nucleotides, ribonucleotides, and deoxyribonucleotides. Incorporation of modified nucleotides into one or both strands of a double-stranded RNA molecule can improve the in vivo stability of the RNA molecule, for example, by reducing the molecule's susceptibility to nucleases and other degradation processes. Incorporation of modified nucleotides can also enhance the efficacy of the RNAi construct for reducing the expression of a target gene.

[0037] In certain embodiments, modified nucleotides have modifications of the ribose sugar. These sugar modifications may include modifications at the 2' and / or 5' positions of the pentose ring, as well as bicyclic sugar modifications. 2'-modified nucleotides refer to nucleotides having a pentose ring with a substituent at the 2' position other than H or OH. Such 2' modifications include, but are not limited to, 2'-O-alkyl (e.g., O-C1-C10 or O-C1-C10 substituted alkyl), 2'-O-allyl (O-CH2CH=CH2), 2'-C-allyl, 2'-fluoro, 2'-O-methyl (OCH3), 2'-O-methoxyethyl (O-(CH2)2OCH3), 2'-OCF3, 2'-O(CH2)2SCH3, 2'-O-aminoalkyl, 2'-amino (e.g., NH2), 2'-O-ethylamine, and 2'-azido. Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy.

[0038] "Bicyclic sugar modification" refers to a modification of the pentose ring in which a bridge connects two atoms of the ring to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides containing a sugar moiety having a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4′-CH2-O-2′) bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4′-CH2-O-2′) BNAs (also referred to as locked nucleic acids or LNAs); ethyleneoxy (4′-(CH2)2-O-2′) BNAs; aminooxy (4′-CH2-ON(R)-2′) BNAs; oxyamino (4′-CH2-N(R)-O-2′) BNAs; methyl(methyleneoxy) (4′-CH(CH3)-O-2′) BNAs (constrained methylene-thio (4'-CH2-S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA; propylene carbocyclic (4'-(CH2)3-2') BNA; and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that may be incorporated into the RNAi constructs of the invention are described in U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, which are incorporated by reference in their entireties.

[0039] In some embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), glycol nucleic acids, or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In a specific embodiment, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0040] Both the sense strand and the antisense strand of an RNAi construct may contain one or more modified nucleotides. For example, in some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides may be 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or a combination thereof.

[0041] In some embodiments, all pyrimidine nucleotides preceding an adenosine nucleotide in the sense strand, the antisense strand, or both strands are modified nucleotides. For example, when the sequence 5'-CA-3' or 5'-UA-3' appears in either strand, the cytidine and uridine nucleotides are modified nucleotides, preferably 2'-O-methyl modified nucleotides. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides), and all 5' nucleotides of the sequence 5'-CA-3' or 5'-UA-3' present in the antisense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides). In other embodiments, all nucleotides in the duplex region are modified nucleotides. In such embodiments, the modified nucleotides are preferably 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

[0042] In embodiments in which the RNAi construct comprises a nucleotide overhang, the nucleotide in the overhang may be a ribonucleotide, a deoxyribonucleotide, or a modified nucleotide. In one embodiment, the nucleotide in the overhang is a deoxyribonucleotide, such as deoxythymidine. In another embodiment, the nucleotide in the overhang is a modified nucleotide. For example, in some embodiments, the nucleotide in the overhang is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-methoxyethyl modified nucleotide, or a combination thereof.

[0043] The RNAi constructs of the present invention may also contain one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage other than the naturally occurring 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiral phosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, modified internucleotide linkages are non-phosphorus-containing internucleotide linkages, and may therefore be referred to as modified internucleoside linkages. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (-O-Si(H)-O-); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (-CH-N(CH)-O-CH-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S, and CH component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to produce peptide nucleic acids or PNAs, such as those described in U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be used in the RNAi constructs of the present invention are described in U.S. Pat. No. 6,693,187, U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entirety.

[0044] In certain embodiments, an RNAi construct comprises one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages may be present in the sense strand, the antisense strand, or both strands of the RNAi construct. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In still other embodiments, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. The RNAi construct may comprise one or more phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 3'-end of the sense strand, the antisense strand, or both strands. In other embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands. In one embodiment, the RNAi construct comprises a single phosphorothioate internucleotide linkage at the 3'-end of the sense strand and a single phosphorothioate internucleotide linkage at the 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3'-end of the antisense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 3'-end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3' and 5' ends of the antisense strand.In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3'- and 5'-ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3'- and 5'-ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at both the 3'- and 5'-ends of the sense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5'- and 3'-ends of the antisense strand, and phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5'- and 3'-ends of the sense strand). In any of the embodiments in which one or both strands comprise one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages within the strands may be native 3'-5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0045] In embodiments in which the RNAi construct comprises a nucleotide overhang, two or more of the unpaired nucleotides in the overhang may be linked by phosphorothioate internucleotide bonds. In certain embodiments, all unpaired nucleotides in the 3'-end nucleotide overhang of the antisense strand and / or sense strand are linked by phosphorothioate internucleotide bonds. In other embodiments, all unpaired nucleotides in the 5'-end nucleotide overhang of the antisense strand and / or sense strand are linked by phosphorothioate internucleotide bonds. In still other embodiments, all unpaired nucleotides in any nucleotide overhang are linked by phosphorothioate internucleotide bonds.

[0046] In certain embodiments, modified nucleotides incorporated into one or both strands of an RNAi construct of the invention have a nucleobase (also referred to herein as a "base") modification. A "modified nucleobase" or "modified base" refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases may be synthetic or naturally occurring modifications, including, but not limited to, universal bases, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thiazolinone ... hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine, and abasic residues (apurinic / apyrimidinic residues lacking a purine or pyrimidine base and lacking a nucleobase at position 1 of the ribose sugar), and inverted nucleotides (nucleotides having a 3'-3' linkage, which may be any of the inverted nucleotides described above, including inverted abasic nucleotides and inverted deoxynucleotides).

[0047] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately forms base pairs with all of the naturally occurring bases in RNA and DNA without altering the resulting double-stranded region's double helix structure. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl, and other aromatic derivatives, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0048] Other suitable modified bases that can be incorporated into the RNAi construct of the present invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol.10:297-310, 2000 and Peacock et al., J.Org.Chern., Vol.76:7295-7300, 2011, both of which are incorporated herein by reference in their entirety.Those skilled in the art will be well aware that guanine, cytosine, adenine, thymine, and uracil can be substituted with other nucleobases, such as the modified nucleobases described above, without substantially changing the base pairing properties of polynucleotides comprising nucleotides having such substituted nucleobases.

[0049] In some embodiments of the RNAi constructs of the present invention, the 5'-end of the sense strand, the antisense strand, or both the antisense strand and the sense strand comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to a terminal phosphate group, including unmodified phosphate (-OP=O)(OH)OH) and modified phosphate. Modified phosphates include phosphates in which one or more of the O and OH groups are replaced with H, O, S, N(R), or alkyl, where R is H, an amino-protecting group, or unsubstituted or substituted alkyl. Exemplary phosphate moieties include, but are not limited to, 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or unmethylated); 5'-adenosine cap or any other modified or unmodified nucleotide cap structure; 5'-monothiophosphate (phosphorothioate); 5'-monodithiophosphate (phosphorodithioate); 5'-alpha-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate; 5'-vinylphosphate; 5'-alkylphosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0050] Modified nucleotides that can be incorporated into RNAi constructs of the present invention may have multiple chemical modifications described herein. For example, modified nucleotides may have a modification to the ribose sugar and a modification to the nucleobase. For example, modified nucleotides may include a 2' sugar modification (e.g., 2'-fluoro or 2'-methyl) and a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, modified nucleotides may include a sugar modification in combination with a modification to the 5' phosphate, which results in a modified internucleotide or internucleoside linkage when the modified nucleotide is incorporated into a polynucleotide. For example, in some embodiments, modified nucleotides may include a sugar modification, such as a 2'-fluoro modification, a 2'-O-methyl modification, or a bicyclic sugar modification, and a 5' phosphorothioate group. Thus, in some embodiments, one or both strands of an RNAi construct of the present invention include a combination of 2'-modified nucleotides or BNAs and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of an RNAi construct of the invention comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs comprising modified nucleotides and internucleotide linkages are shown in Table 2.

[0051] Function of RNAi constructs Preferably, the RNAi constructs of the present invention reduce or inhibit the expression of SCAP in cells, particularly hepatocytes. Thus, in one embodiment, the present invention provides a method for reducing SCAP expression in cells by contacting the cells with any of the RNAi constructs described herein. The cells may be in vitro or in vivo. SCAP expression can be assessed by measuring the amount or level of SCAP mRNA, SCAP protein, or another biomarker associated with SCAP expression. Reduction of SCAP expression in cells or animals treated with the RNAi constructs of the present invention can be determined compared to SCAP expression in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, reduction of SCAP expression is assessed by (a) measuring the amount or level of SCAP mRNA in hepatocytes treated with an RNAi construct of the present invention, (b) measuring the amount or level of SCAP mRNA in hepatocytes treated with a control RNAi construct (e.g., an RNAi construct directed against an RNA molecule not expressed in hepatocytes, or an RNAi construct having a nonsense or scrambled sequence) or without the construct, and (c) comparing the measured SCAP mRNA level from the cells treated in (a) with the measured SCAP mRNA level from the control cells in (b). SCAP mRNA levels in the treated and control cells may be normalized with respect to RNA levels for a control gene (e.g., 18S ribosomal RNA) before comparison. SCAP mRNA levels can be measured by various methods, including Northern blot analysis, nuclease protection assay, fluorescent in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, and quantitative PCR.

[0052] In another embodiment, reduction of SCAP expression is assessed by (a) measuring the amount or level of SCAP protein in hepatocytes treated with an RNAi construct of the present invention, (b) measuring the amount or level of SCAP protein in hepatocytes treated with a control RNAi construct (e.g., an RNAi construct directed against an RNA molecule not expressed in hepatocytes, or an RNAi construct having a nonsense or scrambled sequence) or without the construct, and (c) comparing the measured SCAP protein level from the cells treated in (a) with the measured SCAP protein level from the control cells in (b). Methods for measuring SCAP protein levels are known to those skilled in the art and include Western blot, immunoassays (e.g., ELISA), and flow cytometry. Example 3 describes an exemplary method for measuring SCAP mRNA using RNA FISH. Any method capable of measuring SCAP mRNA or protein can be used to evaluate the effectiveness of an RNAi construct of the present invention.

[0053] In some embodiments, the method for assessing SCAP expression levels is performed in vitro in cells that naturally express SCAP (e.g., hepatocytes) or cells engineered to express SCAP. In certain embodiments, the method is performed in vitro in hepatocytes. Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., human, non-human primate, or rodent hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells.

[0054] In another embodiment, the method for assessing SCAP expression levels is performed in vivo. The RNAi construct and any control RNAi constructs can be administered to an animal (e.g., a rodent or non-human primate), and SCAP mRNA or protein levels can be assessed in liver tissue harvested from the treated animal. Alternatively, or in addition, biomarkers or functional phenotypes associated with SCAP expression can be assessed in the treated animal.

[0055] In certain embodiments, SCAP expression is reduced in hepatocytes by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by an RNAi construct of the present invention. In some embodiments, SCAP expression is reduced in hepatocytes by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by an RNAi construct of the present invention. In other embodiments, SCAP expression is reduced in hepatocytes by about 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, by an RNAi construct of the present invention. The percentage reduction in SCAP expression can be measured by any of the methods described herein and other methods known in the art. For example, in certain embodiments, an RNAi construct of the invention inhibits SCAP expression by at least 45% in Hep3B cells (containing wild-type SCAP) in vitro, as described in Examples 2 and 4. In related embodiments, an RNAi construct of the invention inhibits SCAP expression by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in Hep3B cells in vitro, as described in Examples 2 and 4. In other embodiments, an RNAi construct of the invention inhibits SCAP expression by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% in Hep3B cells in vitro, as described in Examples 2 and 4. In certain embodiments, an RNAi construct of the invention inhibits SCAP expression by at least 45% in C57B16 mouse liver, as described in the Examples. In related embodiments, the RNAi constructs of the present invention inhibit SCAP expression by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in C57B16 mouse liver, as described in the Examples.In other embodiments, the RNAi constructs of the invention inhibit SCAP expression by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% in C57B16 mouse liver, as described in the Examples. SCAP reduction can be measured using various techniques, including RNA FISH or droplet digital PCR, as described in Examples 2 and 4, or in vitro studies, as described in Examples 3, 5, 6, 7, and 8.

[0056] In some embodiments, the efficacy of the RNAi constructs of the present invention in inhibiting SCAP expression in hepatocytes is evaluated by calculating IC50 values. An "IC50 value" is the dose / concentration required to achieve 50% inhibition of biological or biochemical function. The IC50 value of any particular substance or antagonist can be determined by constructing a dose-response curve and testing the effect of different concentrations of the substance or antagonist on expression levels or functional activity in any assay. The IC50 value for a given antagonist or substance can be calculated by determining the concentration required to inhibit half of the maximum biological response or native expression level. Thus, the IC50 value for any RNAi construct can be calculated by determining the concentration of the RNAi construct required to inhibit half of the native SCAP expression level in hepatocytes (e.g., the SCAP expression level in control hepatocytes) in any assay, such as the immunoassay, RNA FISH assay, or droplet digital PCR assay described in the Examples. The RNAi constructs of the present invention can inhibit SCAP expression in hepatocytes (e.g., Hep3B cells) with an IC50 of less than about 100 nM. For example, the RNAi constructs inhibit SCAP expression in hepatocytes with an IC50 of about 0.001 nM to about 100 nM, about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi constructs inhibit SCAP expression in hepatocytes (e.g., Hep3B cells) with an IC50 of about 1 nM to about 10 nM. In certain embodiments, the RNAi constructs inhibit SCAP expression in hepatocytes (e.g., Hep3B cells) with an IC50 of about 0.1 nM to about 5 nM. The RNAi constructs of the present invention can inhibit SCAP expression in hepatocytes (e.g., Hep3B cells) with an IC50 of less than about 20 nM.For example, the RNAi construct inhibits SCAP expression in hepatocytes with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits SCAP expression in hepatocytes (e.g., Hep3B cells) with an IC50 of about 1 nM to about 10 nM.

[0057] In some embodiments, RNAi constructs of the invention can provide long-term in vivo SCAP silencing, for example, in ob / ob mice as described in Example 8. In some embodiments, RNAi constructs of the invention can silence at least 50%, at least 70%, or at least 80% of SCAP expression 20 days after administration of the construct in ob / ob mice as described in Example 8. In some embodiments, RNAi constructs of the invention can silence at least 50%, at least 60%, or at least 70% of SCAP expression 30 days after administration of the construct in ob / ob mice as described in Example 8.

[0058] The RNAi construct of the present invention can be easily produced using techniques known in the art, for example, by conventional solid-phase nucleic acid synthesis.The polynucleotide of the RNAi construct can be constructed using a suitable nucleic acid synthesizer that utilizes standard nucleotide or nucleoside precursors (e.g., phosphoramidites).Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA).

[0059] Oligonucleotides can be synthesized via phosphoramidite chemistry using a 2' silyl protecting group with acid-labile dimethoxytrityl (DMT) at the 5' position of the ribonucleoside. Final deprotection conditions are known not to significantly degrade the RNA product. All syntheses can be performed on any automated or manual synthesizer, on large, medium, or small scales. Synthesis can also be performed in multiwell plates, columns, or glass slides.

[0060] The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluoride. Crown ether catalysts can also be used in combination with inorganic fluorides in the deprotection reaction. Preferred fluoride ion sources are tetrabutylammonium fluoride or amine hydrofluorides (e.g., aqueous HF combined with triethylamine in a dipolar aprotic solvent, such as dimethylformamide).

[0061] The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters to fluoride. Methyl protection of the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve process yields.

[0062] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in the RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, e.g., one that is stable to treatment with acid. Silyl protecting groups meet this requirement and can be easily removed in a final fluoride deprotection step, resulting in minimal RNA degradation.

[0063] Tetrazole catalysts may be used in standard phosphoramidite coupling reactions. Preferred catalysts include tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0064] As can be appreciated by those of skill in the art, additional methods of synthesizing the RNAi constructs described herein will be apparent to those of skill in the art. In addition, the various synthetic steps may be performed in an alternate sequence or order to arrive at the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on bases), and protecting group methodologies (protection and deprotection) useful in synthesizing the RNAi constructs described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Custom synthesis of RNAi constructs is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0065] The RNAi construct of the present invention may contain a ligand. As used herein, "ligand" refers to any compound or molecule that can directly or indirectly interact with another compound or molecule. The interaction of a ligand with another compound or molecule may induce a biological response (e.g., trigger a signal transduction cascade, induce receptor-mediated endocytosis), or may simply be a physical association. A ligand can modify one or more properties of the double-stranded RNA molecule to which it is bound, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties of the RNA molecule.

[0066] The ligand may comprise a serum protein (e.g., human serum albumin, low-density lipoprotein, globulin), a cholesterol moiety, a vitamin (biotin, vitamin E, vitamin B12), a folate moiety, a steroid, a bile acid (e.g., cholic acid), a fatty acid (e.g., palmitic acid, myristic acid), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), a glycoside, a phospholipid, or an antibody or binding fragment thereof (e.g., an antibody or binding fragment that targets the RNAi construct to a specific cell type, such as the liver). Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, lipophilic molecules such as adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, and hexadecylglycerol. , borneol, menthol, 1,3-propanediol, heptadecyl group, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenoic acid, dimethoxytrityl or phenoxazine), peptides (e.g., antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0067] In certain embodiments, the ligand has endosomolytic properties. The endosomolytic ligand promotes lysis of endosomes and / or transport of the RNAi construct of the present invention or its components from endosomes to the cytoplasm of a cell. The endosomolytic ligand can be a polycationic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand adopts its active conformation at endosomal pH. The "active" conformation is one in which the endosomolytic ligand promotes lysis of endosomes and / or transport of the RNAi construct of the present invention or its components from endosomes to the cytoplasm of a cell. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, Vol. 26:2964-2972, 1987), EALA peptide (Vogel et al., J. Am. Chern. Soc., Vol. 118:1581-1586, 1996), and their derivatives (Turk et al., Biochem. Biophys. Acta, Vol. 1559:56-68, 2002). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0068] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12:103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules are also described in U.S. Pat. Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are incorporated herein by reference in their entireties. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated with a folate moiety can be taken up by cells via receptor-mediated endocytosis. Such folate-polynucleotide conjugates are described in U.S. Pat. No. 8,188,247, which is incorporated herein by reference in its entirety.

[0069] Given that SCAP is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to deliver an RNAi construct specifically to such liver cells. In some embodiments, an RNAi construct can be specifically targeted to the liver by employing a ligand that binds to or interacts with a protein expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand may comprise an antigen-binding protein (e.g., an antibody or binding fragment thereof (e.g., Fab, scFv)) that specifically binds to a receptor expressed on hepatocytes.

[0070] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound having at least six carbon atoms (which may be linear, branched, or cyclic) and composed of one or more monosaccharide units, each of which has an oxygen, nitrogen, or sulfur atom attached to it. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from pentose, hexose, or heptose, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0071] In some embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In certain embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective in targeting compounds to hepatocytes. See, e.g., D'Souza and Devarajan, J. Control Release, Vol. 203:126-139, 2015. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs of the invention are described in U.S. Pat. Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Application Publication No. 20030130186; and WO 2013166155, all of which are incorporated by reference herein in their entireties.

[0072] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety containing two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety contains two or more carbohydrate binding domains that can bind to two or more different molecules or to two or more different sites on the same molecule. The valency of a carbohydrate moiety refers to the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" with respect to a carbohydrate moiety refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A multivalent carbohydrate moiety can comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the polyvalent carbohydrate moiety is divalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety can be di- or tri-stranded. In a specific embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another specific embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Exemplary trivalent or tetravalent GalNAc-containing ligands for incorporation into the RNAi constructs of the invention are detailed below.

[0073] The ligand may be directly or indirectly bound or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is directly covalently bound to the sense strand or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently bound to the sense strand or antisense strand of the RNAi construct via a linker. The ligand may be bound to the nucleobase, sugar moiety, or internucleotide bond of the polynucleotide (e.g., the sense strand or antisense strand) of the RNAi construct of the present invention. Conjugation or binding to a purine nucleobase or a derivative thereof may occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is bound to the ligand. Conjugation or binding to a pyrimidine nucleobase or a derivative thereof may also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase may be bound to the ligand. Conjugation or binding to the sugar moiety of a nucleotide can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be bound to a ligand include the 2', 3', and 5' carbon atoms. In basic residues, the 1' position can also be bound to a ligand. The internucleotide bond can also facilitate ligand binding. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand can be bound directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the ligand can be bound to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0074] In certain embodiments, the ligand may be attached to the 3' or 5' end of either the sense strand or the antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3' position of the 3'-terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand but one or more terminal nucleotides before (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2' position of the sugar of the 3'-terminal nucleotide of the sense strand.

[0075] In certain embodiments, the ligand is attached to the sense strand or antisense strand via a linker. A "linker" is an atom or group of atoms that covalently attaches the ligand to the polynucleotide component of the RNAi construct. Linkers can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker may comprise a bifunctional linking moiety, typically comprising an alkyl moiety bearing two functional groups. One of the functional groups is selected to bind to a compound of interest (e.g., the sense strand or antisense strand of the RNAi construct), and the other is selected to essentially bind to any selected group, such as a ligand, as described herein. In certain embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol units or amino acid units. Examples of functional groups typically employed in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups, and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, and unsaturation (e.g., double or triple bonds).

[0076] Linkers that can be used to attach a ligand to the sense or antisense strand in an RNAi construct of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0077] In certain embodiments, the linker is cleavable. A cleavable linker is one that is sufficiently stable outside the cell but is cleaved upon entry into the target cell to release the two moieties that the linker holds together. In some embodiments, the cleavable linker is cleaved at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold or more, or at least 100-fold faster in the target cell or under a first reference condition (e.g., which may be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (e.g., which may be selected to mimic or represent conditions found in blood or serum).

[0078] Cleavable linkers are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are found to be more prevalent or at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include oxidizing enzymes or reductases or reducing agents, such as mercaptans, that are present inside cells and can degrade redox-cleavable linkers by reduction, redox agents that are selective for specific substrates or have no substrate specificity; esterases; agents that can create endosomes or acidic environments, such as those that create a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0079] Cleavable linkers can include a moiety that is sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. The pH of endosomes is more acidic, ranging from 5.5 to 6.0, and the pH of lysosomes is even more acidic, at approximately 5.0. Some linkers have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand into the interior of the cell or into a desired compartment of the cell.

[0080] The linker may contain a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the cells to be targeted. For example, a liver-targeting ligand can be linked to an RNA molecule via a linker containing an ester group. Because liver cells are rich in esterases, the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other types of cells that are rich in esterases include lung, renal cortex, and testicular cells. Linkers containing peptide bonds can be used when targeting cells rich in peptidases, such as liver cells and synovial cells.

[0081] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition selected to be indicative of cleavage in target cells and a second condition selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell cultures, organs, or tissue cultures, or in whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm them with further evaluations in whole animals. In some embodiments, useful candidate linkers are cleaved at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0082] In other embodiments, a redox-cleavable linker is utilized. A redox-cleavable linker is cleaved upon reduction or oxidation. An example of a reductively cleavable group is a disulfide bond (-SS-). One or more methods described herein can be used to determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or is suitable for use with, for example, a particular RNAi construct and a particular ligand. For example, a candidate linker can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate that would be observed in cells, e.g., target cells. Alternatively, the candidate linker can be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, the candidate linker is cleaved at a rate of up to 10% in blood. In other embodiments, useful linker candidates are degraded at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

[0083] In yet another embodiment, the phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that hydrolyzes phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based cleavable groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Particular embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. Another particular embodiment is -OP(O)(OH)-O-. These linker candidates can be evaluated using methods similar to those described above.

[0084] In other embodiments, the linker may include an acid-cleavable group, which is a group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable group is cleaved in an acidic environment of about pH 6.5 or below (e.g., about 6.0, 5.5, 5.0 or below) or by an agent, such as an enzyme, that can act as a general acid. Within cells, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). In a particular embodiment, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethyl, pentyl, or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0085] In other embodiments, the linker may include an ester-based cleavable group, which is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable groups have the general formula -C(O)O- or -OC(O)-. These linker candidates can be evaluated using methods similar to those described above.

[0086] In further embodiments, the linker may comprise a peptide-based cleavable group, which is cleaved by enzymes such as intracellular peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. Such candidates can be evaluated using methods similar to those described above.

[0087] Other types of linkers suitable for attaching a ligand to the sense or antisense strand in the RNAi constructs of the present invention are known in the art and may include the linkers described in U.S. Pat. Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are incorporated herein by reference in their entirety.

[0088] In certain embodiments, the ligand covalently attached to the sense strand or antisense strand of an RNAi construct of the invention comprises a GalNAc moiety, e.g., a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3'-end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3'-end of the sense strand. In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5'-end of the sense strand. In some embodiments, the GalNAc moiety is attached to the 5'-end of the sense strand of an odd-numbered sequence selected from SEQ ID NOs: 1-159, 161-319, 321-461, or 463-603.

[0089] In some embodiments, the RNAi constructs of the present invention may be delivered to cells or tissues of interest by administering a vector that encodes and controls the intracellular expression of the RNAi construct. A "vector" (also referred to herein as an "expression vector") is a composition that can be used to deliver a nucleic acid of interest inside a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors. Vectors may replicate within living cells or may be synthetically produced.

[0090] Generally, vectors for expressing RNAi constructs of the present invention will contain one or more promoters operably linked to a sequence encoding the RNAi construct. As used herein, the phrases "operably linked" or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to a polynucleotide sequence to control initiation of transcription by RNA polymerase and expression of the polynucleotide sequence. A "promoter" refers to a sequence recognized by the cellular or introduced synthetic machinery required to initiate specific transcription of a gene sequence. Suitable promoters include, but are not limited to, RNA pol I, pol II, H1, or U6 RNA pol III, and viral promoters (e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In some embodiments, the H1 or U6 RNA pol III promoter is preferred. The promoter may be a tissue-specific or inducible promoter. Of particular interest are liver-specific promoters, such as promoter sequences from the human α1-antitrypsin gene, albumin gene, hemopexin gene, and hepatic lipase gene. Inducible promoters include promoters regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-PD1-thiogalactopyranoside (IPTG).

[0091] In some embodiments in which the RNAi construct comprises an siRNA, the two separate strands (sense and antisense strands) may be expressed from a single vector or from two separate vectors. For example, in one embodiment, the sequence encoding the sense strand is operably linked to a promoter on a first vector, and the sequence encoding the antisense strand is operably linked to a promoter on a second vector. In such embodiments, the first and second vectors are simultaneously introduced into a target cell, e.g., by infection or transfection, so that the sense and antisense strands are transcribed and hybridize to form an siRNA molecule within the cell. In another embodiment, the sense and antisense strands are transcribed from two separate promoters located in a single vector. In some such embodiments, the sequence encoding the sense strand is operably linked to a first promoter, and the sequence encoding the antisense strand is operably linked to a second promoter, and the first and second promoters are located in a single vector. In one embodiment, the vector comprises a first promoter operably linked to a sequence encoding an siRNA molecule and a second promoter operably linked in the opposite direction to the same sequence, such that transcription of the sequence from the first promoter results in synthesis of the sense strand of the siRNA molecule, and transcription of the sequence from the second promoter results in synthesis of the antisense strand of the siRNA molecule.

[0092] In other embodiments, the RNAi construct comprises an shRNA, wherein a sequence encoding a single, at least partially self-complementary RNA molecule is operably linked to a promoter that generates a single transcript. In some embodiments, the sequence encoding the shRNA comprises inverted repeats connected by a linker polynucleotide sequence to generate the stem and loop structure of the shRNA after transcription.

[0093] In some embodiments, the vector encoding the RNAi construct of the present invention is a viral vector.Various viral vector systems suitable for expressing the RNAi construct described herein include but are not limited to adenovirus vector, retrovirus vector (for example, lentivirus vector, Moloney murine leukemia virus), adeno-associated virus vector; herpes simplex virus vector; SV40 vector; polyomavirus vector; papillomavirus vector; picornavirus vector; and poxvirus vector (for example, vaccinia virus).In certain embodiments, the viral vector is a retrovirus vector (for example, lentivirus vector).

[0094] Various vectors suitable for use in the present invention, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into the vectors, and methods for delivering the vectors to cells of interest are within the skill of one in the art. For example, Dornburg, Gene Therap.,Vol.2:301-310,1995;Eglitis,Biotechniques,Vol.6:608-614,1988;Miller,HumGene Therap.,Vol.1:5-14,1990;Anderson,Nature,Vol.392:25-30,1998;Rubinson DA et al.,Nat. Genet.,Vol.33:401-406,2003;Brummelkamp et al.,Science,Vol.296:550-553,2002;Brummelkamp et al.,Cancer Cell,Vol.2:243-247,2002;Lee et al.,Nat Biotechnol,Vol.20:500-505,2002;Miyagishi et al., Nat Biotechnol, Vol. 20:497-500, 2002; Paddison et al., GenesDev, Vol. 16:948-958, 2002; Paul et al., Nat Biotechnol, Vol. 20:505-508, 2002; Sui et al., Proc Natl Acad Sci USA, Vol. 99:5515-5520, 2002; and Yu et al., Proc Natl Acad Sci USA, Vol. 99:6047-6052, 2002 (all of which references are incorporated herein by reference in their entireties).

[0095] The present invention also includes pharmaceutical compositions and formulations comprising the RNAi constructs described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions and formulations are useful for reducing SCAP expression in subjects in need thereof. For clinical use, pharmaceutical compositions and formulations will be prepared in a form appropriate for the intended use. Generally, this will involve preparing compositions that are essentially free of pyrogens and other impurities that may be harmful to humans or animals.

[0096] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers, excipients, or diluents" include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are acceptable for use in formulating a drug, such as a drug suitable for human administration. The use of such media and agents with pharmaceutically active substances is well known in the art. Any conventional media or agent is contemplated for use in therapeutic compositions, except insofar as it is incompatible with the RNAi construct of the present invention. Supplementary active ingredients may also be incorporated into the compositions, provided that they do not inactivate the vector or RNAi construct of the composition.

[0097] The compositions and methods for formulating pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the type and severity of the disease or disorder to be treated, or the dose to be administered. In some embodiments, pharmaceutical compositions are formulated based on the intended delivery route. For example, in certain embodiments, pharmaceutical compositions are formulated for parenteral delivery. Parenteral delivery forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (e.g., a GalNAc-containing ligand described herein).

[0098] In some embodiments, the pharmaceutical composition comprises an effective amount of the RNAi construct described herein. An "effective amount" is an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, an effective amount is an amount sufficient to reduce SCAP expression in the subject's hepatocytes. In some embodiments, an effective amount may be an amount sufficient to only partially reduce SCAP expression, for example, to a level comparable to the expression of a wild-type SCAP allele in a human heterozygote.

[0099] An effective amount of an RNAi construct of the present invention can be about 0.01 mg / kg to about 100 mg / kg body weight, about 0.05 mg / kg to about 75 mg / kg body weight, about 0.1 mg / kg to about 50 mg / kg body weight, about 1 mg / kg to about 30 mg / kg body weight, about 2.5 mg / kg to about 20 mg / kg body weight, or about 5 mg / kg to about 15 mg / kg body weight. In certain embodiments, a single effective amount of an RNAi construct of the present invention can be about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg. A pharmaceutical composition comprising an effective amount of an RNAi construct may be administered weekly, biweekly, monthly, quarterly, or semi-annually. The precise determination of what will be considered an effective dosage and frequency of administration can be based on several factors, including the size, age, and general condition of the patient, the type of disorder being treated (e.g., myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia), the particular RNAi construct employed, and the route of administration. Estimates of effective dosages and in vivo half-lives for any particular RNAi construct of the invention can be confirmed using conventional methods and / or testing in appropriate animal models.

[0100] The pharmaceutical composition of the present invention can be administered via any common route as long as the target tissue is accessible via that route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or direct injection into liver tissue or delivery via the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0101] Colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the RNAi constructs of the present invention or vectors encoding such constructs. Commercially available fat emulsions suitable for delivering nucleic acids of the present invention include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and other similar fat emulsions. A preferred colloidal system for in vivo use as a delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the present invention may be encapsulated within liposomes or complexed to liposomes, particularly cationic liposomes. Alternatively, the RNAi constructs of the present invention may be complexed to lipids, particularly cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505, 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO 03 / 093449.

[0102] In some embodiments, the RNAi constructs of the present invention are fully encapsulated within a lipid formulation to form, for example, SPLPs, pSPLPs, SNALPs, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLPs. As used herein, the term "SPLP" refers to nucleic acid-lipid particles containing plasmid DNA encapsulated within lipid vesicles. SNALPs and SPLPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs and SPLPs exhibit long circulatory lifetimes after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them highly useful for systemic administration. Examples of SPLPs include "pSPLPs" containing encapsulated condensing agent-nucleic acid complexes, as described in WO 00 / 03683. Nucleic acid-lipid particles typically have an average diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm, and are substantially non-toxic. Additionally, the nucleic acid present in the nucleic acid-lipid particles is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and WO 96 / 40964.

[0103] Examples of pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that they are easily syringable. Preparations should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0104] Sterile injectable solutions can be prepared by incorporating the active compound in an appropriate amount into a solvent with any other desired ingredients (for example, as listed above), and then sterilizing by filtration.Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and any other desired ingredients, for example, as listed above.For sterile powders for preparing sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient and any additional desired ingredients from the solution that has previously been sterile-filtered.

[0105] The compositions of the present invention can generally be formulated in neutral or salt form. Examples of pharmaceutically acceptable salts include acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, and mandelic acid). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, and procaine).

[0106] For parenteral administration via aqueous solutions, for example, the solution is usually suitably buffered and the liquid diluent first rendered isotonic, e.g., with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is employed, as known to those skilled in the art, particularly in light of the present disclosure. By way of example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises or consists of sterile saline and the RNAi construct described herein. In other embodiments, pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet other embodiments, pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and phosphate buffered saline (PBS).

[0107] In some embodiments, the pharmaceutical composition of the present invention is packaged with or stored in an administration device.Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, automatic injectors, injection pumps, wearable injectors, and injection pens.Devices for aerosolized formulations or powder formulations include, but are not limited to, inhalers, inhalers, and inhalers.Therefore, the present invention includes an administration device that comprises the pharmaceutical composition of the present invention for treating or preventing one or more of the disorders described herein.

[0108] Methods for inhibiting SCAP expression The present invention also provides a method for inhibiting expression of the SCAP gene in a cell. The method includes inhibiting expression of SCAP in the cell by contacting the cell with an RNAi construct, e.g., a double-stranded RNAi construct, in an amount effective to inhibit expression of SCAP in the cell. Contacting the cell with the RNAi construct, e.g., a double-stranded RNAi construct, can be performed in vitro or in vivo. Contacting the cell with the RNAi construct in vivo includes contacting a cell or cells in a subject, e.g., a human subject, with the RNAi construct. A combination of in vitro and in vivo cell contacting methods is also possible.

[0109] The present invention provides methods for reducing or inhibiting the expression of SCAP in a subject in need thereof, and methods for treating or preventing a condition, disease, or disorder associated with SCAP expression or activity. A "condition, disease, or disorder associated with SCAP expression" refers to a condition, disease, or disorder in which altered or elevated expression levels of SCAP are associated with an increased risk of developing the condition, disease, or disorder.

[0110] As discussed above, contacting cell can be direct or indirect.In addition, contacting cell can be achieved through targeting ligand, including any ligand described herein or known in the art.In a preferred embodiment, targeting ligand is carbohydrate moiety, for example, GalNAc ligand, trivalent GalNAc moiety, or any other ligand that can direct RNAi construct to the site of interest.

[0111] In one embodiment, contacting a cell with an RNAi construct includes "introducing" or "delivering" the RNAi construct into the cell by promoting or causing uptake or absorption into the cell. Absorption or uptake of the RNAi construct can occur by unassisted diffusive or active cellular processes, or by auxiliary agents or devices. Introducing the RNAi construct into the cell can be in vitro and / or in vivo. For example, for in vivo introduction, the RNAi construct can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below and / or known in the art.

[0112] As used herein, the term "inhibiting" is used interchangeably with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes all levels of inhibition.

[0113] The phrase "inhibiting expression of SCAP" is intended to refer to the inhibition of expression of any SCAP gene (e.g., mouse SCAP gene, rat SCAP gene, monkey SCAP gene, or human SCAP gene) and variants or mutants of the SCAP gene. Thus, the SCAP gene may be a wild-type SCAP gene, a mutant SCAP gene (such as a mutant SCAP gene that causes triglyceride deposition), or a transgenic SCAP gene in the context of a genetically engineered cell, cell population, or organism.

[0114] "Inhibiting SCAP gene expression" includes inhibition of any level of SCAP gene expression, e.g., at least partial suppression of SCAP gene expression. SCAP gene expression can be assessed based on the level or change in level of any variable associated with SCAP gene expression, such as, for example, SCAP mRNA level, SCAP protein level, or the number or extent of triglyceride deposits. The level can be assessed in an individual cell or a group of cells, e.g., a sample derived from a subject.

[0115] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with SCAP expression compared to a control level, which can be any type of control level utilized in the art, such as a baseline level before administration or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control). In some embodiments of the methods of the present invention, expression of the SCAP gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0116] Inhibition of SCAP gene expression can be demonstrated by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) that has been treated such that the SCAP gene is transcribed and expression of the SCAP gene is inhibited (e.g., by contacting the cell with an RNAi construct of the present invention or by administering an RNAi construct of the present invention to a subject in which the cell is or was present), compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been so treated. In a preferred embodiment, inhibition is demonstrated by a reduction in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) that has been treated such that the SCAP gene is transcribed and expression of the SCAP gene is inhibited (e.g., by contacting the cell with an RNAi construct of the present invention or by administering an RNAi construct of the present invention to a subject in which the cell is or was present), compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been so treated.

number

[0117] Alternatively, inhibition of SCAP gene expression can be assessed for reduction of a parameter functionally related to SCAP gene expression, e.g., SCAP protein expression or SREBP pathway protein activity. SCAP gene silencing can be determined in any cell that expresses SCAP, either constitutively or by genomic manipulation, and by any assay known in the art.

[0118] Inhibition of SCAP protein expression can be evidenced by a reduction in the level of SCAP protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for assessment of mRNA suppression, inhibition of protein expression levels in treated cells or groups of cells can similarly be expressed as a percentage of the level of protein in control cells or groups of cells.

[0119] A control cell or group of cells that can be used to evaluate inhibition of SCAP gene expression includes a cell or group of cells that has not yet been contacted with an RNAi construct of the present invention. For example, a control cell or group of cells can be obtained from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi construct.

[0120] The level of SCAP mRNA expressed by a cell or group of cells, or the level of circulating SCAP mRNA, can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of SCAP expression in a sample is determined by detecting a transcribed polynucleotide or a portion thereof, such as the mRNA of the SCAP gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating SCAP mRNA can be detected using the methods described in PCT / US2012 / 043584, the entire contents of which are incorporated herein by reference.

[0121] In one embodiment, the level of SCAP expression is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific SCAP. Probes can be synthesized by those skilled in the art or obtained from appropriate biological preparations. Probes may also be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0122] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to SCAP mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, e.g., by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, e.g., in an Affymetrix gene chip array. One skilled in the art can easily adapt known mRNA detection methods for use in determining SCAP mRNA levels.

[0123] Alternative methods for determining the level of SCAP expression in a sample include, for example, RT-PCR (experimental embodiment described in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. (1988) Bio / Technology 6:1197), and the like. al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, for example, of mRNA in a sample through the process of nucleic acid amplification and / or reverse transcriptase (to prepare cDNA), followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are useful for detecting nucleic acid molecules, particularly if they are present in very low numbers. In certain embodiments of the present invention, the level of SCAP expression is determined by quantitative fluorescent RT-PCR (i.e., TaqMan™ System). The expression level of SCAP mRNA can be monitored using membrane blots (such as those used in hybridization analyses such as Northern and dot), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining the level of SCAP expression may involve the use of a nucleic acid probe in solution.

[0124] In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of these methods is described and illustrated in the Examples presented herein.

[0125] The level of SCAP protein expression can be determined using any method known in the art for measuring protein levels, including electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high-diffusion chromatography, liquid or gel precipitation, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or dual), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and electrochemiluminescence assay.

[0126] In some embodiments, the effectiveness of the methods of the present invention can be monitored by detecting or monitoring a reduction in symptoms of SCAP disease, such as reduced edematous swelling of the extremities, face, larynx, upper airway, abdomen, trunk, and genitals, prodromal symptoms, laryngeal edema, non-pruritic rash, nausea, vomiting, or abdominal pain. These symptoms can be assessed in vitro or in vivo using any method known in the art.

[0127] In some embodiments of the methods of the present invention, an RNAi construct is administered to a subject such that the RNAi construct is delivered to a specific site within the subject. Inhibition of SCAP expression can be assessed by measuring the level or change in the level of SCAP mRNA or SCAP protein in a sample derived from a body fluid or tissue from a specific site within the subject. In a preferred embodiment, the site is selected from the group consisting of the liver, choroid plexus, retina, and pancreas. The site may also be a small unit or subgroup of cells from any one of the aforementioned sites. The site may also include cells expressing a specific type of receptor.

[0128] Methods for treating or preventing SCAP-associated diseases The present invention provides therapeutic and preventive methods, including administering a composition comprising an RNAi construct of the present invention, a pharmaceutical composition comprising an RNAi construct, or a vector comprising an RNAi construct to a subject suffering from or susceptible to a SCAP-related disease, disorder, and / or symptom. Non-limiting examples of SCAP-related diseases include fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, liver inflammation, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia, or non-alcoholic fatty liver disease (NAFLD). In one embodiment, the SCAP-related disease is NAFLD. In another embodiment, the SCAP-related disease is NASH. In another embodiment, the SCAP-related disease is fatty liver (steatosis). In another embodiment, the SCAP-related disease is insulin resistance. In another embodiment, the SCAP-related disease is not insulin resistance. In some embodiments, SCAP RNAi can be used to treat hepatocellular carcinoma. Increased SREBP activity has been documented in human HCC samples, and evidence points to its causative role in HCC growth. SCAP RNAi (e.g., siRNA) treatment in rodent models of HCC (e.g., xenograft transplantation of HCC cells or liver expression of oncogenes) can result in a reduction in liver tumor burden (i.e., tumor volume).

[0129] In certain embodiments, the present invention provides a method for reducing SCAP expression in a patient in need thereof, comprising administering any of the RNAi constructs described herein to the patient. As used herein, the term "patient" refers to a mammal, including humans, and can be used interchangeably with the term "subject." Preferably, the expression level of SCAP in the patient's hepatocytes is reduced after administration of the RNAi construct, compared to the expression level of SCAP in the patient who has not received the RNAi construct.

[0130] The methods of the present invention are useful for treating subjects suffering from SCAP-related diseases, such as subjects who would benefit from reducing SCAP gene expression and / or SCAP protein production. In one embodiment, the present invention provides a method for reducing the level of SREBP cleavage-activating protein (SCAP) gene expression in a subject suffering from non-alcoholic fatty liver disease (NAFLD). In another embodiment, the present invention provides a method for reducing the level of SCAP protein in a subject suffering from NAFLD. The present invention also provides a method for reducing the level of Hedgehog pathway activity in a subject suffering from NAFLD.

[0131] In another aspect, the present invention provides a method for treating a subject suffering from NAFLD. In one aspect, the present invention provides a method for treating a subject suffering from a SCAP-related disease, such as fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, liver inflammation, hepatocellular necrosis, liver fibrosis, obesity, hepatocellular carcinoma, myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia, or non-alcoholic fatty liver disease (NAFLD). The treatment method (and use) of the present invention comprises administering to a subject, e.g., a human, a therapeutically effective amount of an RNAi construct of the present invention targeting the SCAP gene, a pharmaceutical composition comprising an RNAi construct of the present invention targeting the SCAP gene, or a vector of the present invention comprising an RNAi construct targeting the SCAP gene.

[0132] In one aspect, the present invention provides a method for preventing at least one symptom in a subject suffering from NAFLD, such as the presence of an elevated signaling pathway, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider veins, yellowing of the skin and eyes (jaundice), itching, fluid accumulation and swelling of the legs (edema), abdominal distension (ascites), and mental confusion. The method comprises administering a therapeutically effective amount of an RNAi construct, such as a dsRNA, pharmaceutical composition, or vector of the present invention, to the subject, thereby preventing at least one symptom in a subject suffering from a disorder that would benefit from reducing SCAP gene expression.

[0133] In another aspect, the present invention provides use of a therapeutically effective amount of an RNAi construct of the present invention to treat a subject, e.g., a subject that would benefit from reduced and / or inhibited SCAP gene expression. In a further aspect, the present invention provides use of an RNAi construct of the present invention targeting the SCAP gene, e.g., dsRNA, or a pharmaceutical composition comprising an RNAi construct targeting the SCAP gene, in the manufacture of a medicament for treating a subject, such as a subject suffering from a disorder, e.g., a SCAP-associated disease, that would benefit from reduced SCAP gene expression, e.g., a subject that would benefit from reduced and / or inhibited SCAP gene expression and / or SCAP protein production.

[0134] In another aspect, the present invention provides the use of an RNAi, e.g., a dsRNA, of the present invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from the reduction and / or inhibition of SCAP gene expression and / or SCAP protein production.

[0135] In a further aspect, the present invention provides the use of an RNAi construct of the present invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from the reduction and / or inhibition of SCAP gene expression and / or SCAP protein production, such as a SCAP-associated disease.

[0136] In one embodiment, the RNAi construct targeting SCAP is designed to, for example, increase or decrease expression of the SCAP gene in cells, tissues, blood, or other tissue or bodily fluid of a subject by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, , 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more reduction in SCAP-associated disease, such as nonalcoholic fatty liver disease (NAFLD).

[0137] The methods and uses of the present invention include administering a composition described herein such that expression of the target SCAP gene is reduced for, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target SCAP gene is reduced for an extended period of time, e.g., at least about 2, 3, 4, 5, 6, 7 days or more, e.g., about 1 week, 2 weeks, 3 weeks, or about 4 weeks or more.

[0138] Administration of an RNAi construct according to the methods and uses of the present invention can result in a reduction in the severity, signs, symptoms, and / or markers of a SCAP-related disease, such as nonalcoholic fatty liver disease (NAFLD), in patients suffering from such a disease or disorder. "Reduction" in this context refers to a statistically significant decrease in such levels. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. The effectiveness of disease treatment or prevention can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of a medication required to maintain treatment efficacy, the level of a disease marker, or any other measurable parameter appropriate for the given disease being treated or targeted for prevention. It is well within the capabilities of those skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. For example, the effectiveness of a treatment for NAFLD can be evaluated by, for example, periodic monitoring of NAFLD symptoms, liver fat levels, or the expression of downstream genes. Comparison of subsequent readings with the initial reading provides the physician with an indication of whether the treatment is effective. It is well within the capabilities of those skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. In relation to the administration of an RNAi construct targeting SCAP or a pharmaceutical composition thereof, "effective against" a SCAP-related disease means that administration in a clinically relevant manner results in beneficial effects, such as symptom improvement, cure, disease reduction, prolongation of life, improved quality of life, or other effects generally recognized as favorable by physicians familiar with the treatment of NAFLD and / or SCAP-related diseases and related causes, for at least a statistically significant proportion of patients.

[0139] Treatment or prevention is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is a lack of progression or worsening of disease symptoms that would normally be expected.As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in measurable parameters of the disease may indicate effective treatment.The effectiveness of a given RNAi drug or formulation of the drug may also be determined using an experimental animal model for a given disease known in the art.When using an experimental animal model, the effectiveness of treatment is demonstrated when a statistically significant reduction in markers or disease symptoms is observed.

[0140] Administration of an RNAi construct may, for example, increase the presence of SCAP protein levels in a patient's cells, tissues, blood, urine, or other compartment by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, The price may be reduced by 6%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0141] Prior to administration of the full dose of the RNAi construct, the patient may be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as allergic reactions. In another example, the patient may be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0142] Due to the inhibitory effect on SCAP expression, the composition according to the present invention or a pharmaceutical composition prepared therefrom can improve the quality of life.

[0143] The RNAi constructs of the present invention may be administered in a "naked" form, in which case the modified or unmodified RNAi construct is directly suspended in an aqueous or suitable buffer solution as a "free RNAi." The free RNAi is administered in the absence of a pharmaceutical composition.

[0144] RNAi may be in pharmaceutical composition with suitable buffer solution.The buffer solution may contain acetate, citrate, prolamin, carbonate, phosphate, or any combination thereof.In one embodiment, the buffer solution is phosphate buffered saline (PBS).The pH and osmolality of the buffer solution containing RNAi construct may be adjusted to be suitable for administration to a subject.

[0145] Alternatively, the RNAi constructs of the present invention may be administered as pharmaceutical compositions, such as liposomal formulations of the RNAi construct.

[0146] Subjects who would benefit from reducing and / or inhibiting SCAP gene expression are those suffering from non-alcoholic fatty liver disease (NAFLD) and / or a SCAP-related disease or disorder described herein.

[0147] Treatment of subjects that would benefit from reducing and / or inhibiting SCAP gene expression includes therapeutic and prophylactic treatments.

[0148] The present invention further provides methods for treating subjects who would benefit from the reduction and / or inhibition of SCAP gene expression, e.g., subjects suffering from SCAP-associated diseases, and uses of RNAi constructs or pharmaceutical compositions thereof, in combination with other pharmaceuticals and / or other therapies, e.g., known pharmaceuticals and / or known treatments, e.g., those currently used to treat these disorders.

[0149] For example, in certain embodiments, an RNAi construct targeting the SCAP gene is administered in combination with an agent useful for treating a SCAP-related disease, for example, as described elsewhere herein. For example, additional therapeutic agents and therapies suitable for treating a subject who would benefit from reducing SCAP expression, for example, a subject suffering from a SCAP-related disease, include RNAi constructs targeting different portions of the SCAP gene, therapeutic agents and / or procedures for treating a SCAP-related disease, or any combination of the foregoing.

[0150] In certain embodiments, a first RNAi construct targeting the SCAP gene is administered in combination with a second RNAi construct targeting a different portion of the SCAP gene. For example, a first RNAi construct comprises a first sense strand and a first antisense strand forming a duplex region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand being conjugated to a ligand attached at its 3' end, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker; and a second RNAi construct comprises a second sense strand and a second antisense strand forming a duplex region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, the second sense strand being conjugated to a ligand attached at its 3' end, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker.

[0151] In one embodiment, all of the nucleotides of the first and second sense strands and / or all of the nucleotides of the first and second antisense strands comprise the modification.

[0152] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0153] In certain embodiments, the first RNAi construct that targets SCAP gene is administered in combination with the second RNAi construct that targets a gene other than SCAP gene.For example, the RNAi construct that targets SCAP gene can be administered in combination with the RNAi construct that targets SCAP gene.The first RNAi construct that targets SCAP gene and the gene that is other than SCAP gene, for example, the second RNAi construct that targets SCAP gene, can be administered as part of the same pharmaceutical composition.In addition, the first RNAi construct that targets SCAP gene and the gene that is other than SCAP gene, for example, the second RNAi construct that targets SCAP gene, can be administered as part of different pharmaceutical compositions.

[0154] The RNAi construct and additional therapeutic agent and / or treatment may be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agent may be administered as part of a separate composition or separately and / or by other methods known in the art or described herein.

[0155] The present invention also provides methods of using RNAi constructs of the present invention and / or compositions containing RNAi constructs of the present invention to reduce and / or inhibit SCAP expression in cells. In another aspect, the present invention provides RNAi constructs of the present invention and / or compositions containing RNAi constructs of the present invention for use in reducing and / or inhibiting SCAP gene expression in cells. In yet another aspect, the present invention provides use of RNAi constructs of the present invention and / or compositions containing RNAi constructs of the present invention for the manufacture of a medicament for reducing and / or inhibiting SCAP gene expression in cells. In yet another aspect, the present invention provides RNAi constructs of the present invention and / or compositions containing RNAi constructs of the present invention for use in reducing and / or inhibiting SCAP protein production in cells. In yet another aspect, the present invention provides use of RNAi constructs of the present invention and / or compositions containing RNAi constructs of the present invention for the manufacture of a medicament for reducing and / or inhibiting SCAP protein production in cells. The methods and uses include inhibiting expression of the SCAP gene in a cell or inhibiting SCAP protein production by contacting the cell with an RNAi construct of the present invention and maintaining the cell for a period of time sufficient to obtain degradation of the mRNA transcript of the SCAP gene.

[0156] The reduction in gene expression can be evaluated by any method known in the art. For example, the reduction in SCAP expression can be evaluated by determining the mRNA expression level of SCAP using methods conventional to those skilled in the art, such as Northern blotting, qRT-PCR, determining the protein level of SCAP using methods conventional to those skilled in the art, such as Western blotting, immunological techniques, flow cytometry, ELISA, and / or determining the biological activity of SCAP.

[0157] In the methods and uses of the present invention, the cells may be contacted in vitro or in vivo, i.e., the cells may be within a subject.

[0158] Cells suitable for treatment using the methods of the present invention can be any cells that express the SCAP gene, such as cells derived from a subject suffering from NAFLD, or cells containing an expression vector containing the SCAP gene or a portion of the SCAP gene. Cells suitable for use in the methods and uses of the present invention can be mammalian cells, such as primate cells (human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (such as cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), avian cells (such as duck cells or goose cells), or whale cells. In one embodiment, the cells are human cells.

[0159] SCAP gene expression is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 1 The expression level may be inhibited by 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

[0160] SCAP protein production is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 1 , 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% inhibition.

[0161] The in vivo method and use of the present invention may include administering to a subject a composition containing an RNAi construct, wherein the RNAi construct comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the SCAP gene of the mammal to be treated. When the organism to be treated is a human, the composition can be administered by any means known in the art, including, but not limited to, subcutaneous, intravenous, oral, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and parenteral routes, and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection. In one embodiment, the composition is administered by subcutaneous injection.

[0162] In some embodiments, administration is via depot injection. Depot injection can consistently release RNAi over a long period of time. Therefore, depot injection can reduce the frequency of administration required to achieve a desired effect, such as the desired inhibition of SCAP, or a therapeutic or preventive effect. Depot injection can also achieve a more consistent serum concentration. Depot injection can include subcutaneous injection or intramuscular injection. In a preferred embodiment, depot injection is subcutaneous injection.

[0163] In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump implanted subcutaneously. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi construct to a subject.

[0164] The mode of administration may be selected based on whether local or systemic treatment is desired and on the area to be treated. The route and site of administration may be selected to enhance targeting.

[0165] In one embodiment, the present invention also provides a method for inhibiting the expression of the SCAP gene in a mammal, for example, a human. The present invention also provides a composition comprising an RNAi construct targeting the SCAP gene in mammalian cells, which is used to inhibit the expression of the SCAP gene in a mammal. In another embodiment, the present invention provides the use of an RNAi construct targeting the SCAP gene in mammalian cells in the manufacture of a medicament for inhibiting the expression of the SCAP gene in a mammal.

[0166] The methods and uses include administering to a mammal, e.g., a human, a composition comprising an RNAi construct that targets the SCAP gene in the cells of the mammal, and maintaining the mammal for a period of time sufficient to obtain degradation of the mRNA transcripts of the SCAP gene, thereby inhibiting expression of the SCAP gene in the mammal.

[0167] Reduction of gene expression can be assessed in peripheral blood samples from RNAi-treated subjects by any method known in the art, for example, qRT-PCR as described herein. Reduction of protein production can be assessed by any method known in the art, including, for example, ELISA or Western blotting as described herein. In one embodiment, a tissue sample serves as the tissue source for monitoring reduction of SCAP gene and / or protein expression. In another embodiment, a blood sample serves as the tissue source for monitoring reduction of SCAP gene and / or protein expression.

[0168] In one embodiment, verification of RISC-mediated cleavage of the target in vivo after administration of the RNAi construct is performed by performing 5'-RACE or a modification of protocols known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38(3)p-el9) (Zimmermann et al. (2006) Nature 441:111-4).

[0169] It is understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by substituting thymine bases for uracil bases in the sequences. Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by substituting thymine bases for uracil bases in the sequences. Deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing mixtures of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein are included in the present invention.

[0170] Additionally, any nucleic acid sequence disclosed herein may be modified by any combination of chemical modifications. Those skilled in the art will readily understand that, in certain instances, designations such as "RNA" or "DNA" to describe modified polynucleotides are arbitrary. For example, a polynucleotide containing nucleotides having a 2'-OH substituent on the ribose sugar and a thymine base may be described as a DNA molecule with modified sugars (2'-OH for the native 2'-H in DNA) or an RNA molecule with modified bases (thymine (methylated uracil) for the native uracil in RNA).

[0171] Thus, the nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of native or modified RNA and / or DNA, including, but not limited to, those in the sequence listing, including, but not limited to, those with modified nucleic acid bases. As a further example, and without limitation, a polynucleotide having the sequence "ATCGATCG" encompasses all polynucleotides having such sequences, whether modified or unmodified, including, but not limited to, those with RNA bases, e.g., the sequence "AUCGAUCG," as well as compounds including polynucleotides with some DNA bases and some RNA bases, such as "AUCGATCG," and other modified bases, such as "ATmeCGAUCG," where meC indicates a cytosine base with a methyl group at the 5-position.

[0172] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the appended claims.

[0173] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. To the extent that any definitions or terms provided in a reference incorporated by reference differ from the terms and discussion provided herein, the terms and definitions shall control.

[0174] equivalent The foregoing specification is considered sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears, it will be understood that the invention can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0175] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the invention. [Example]

[0176] Example 1: Selection, design, and synthesis of modified SCAP siRNA molecules Identification and selection of optimal sequences for therapeutic siRNA molecules targeting sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) was identified using bioinformatics analysis of the human SCAP transcript (NM_012235). Table 1 shows the sequences identified as having therapeutic properties. Throughout the various sequences, "invAb" is an inverted abasic nucleotide.

[0177] [Table 1]

[0178] [Table 2]

[0179] [Table 3]

[0180] [Table 4]

[0181] [Table 5]

[0182] To improve the potency and in vivo stability of SCAP siRNA sequences, chemical modifications were incorporated into SCAP siRNA molecules. Specifically, 2'-O-methyl and 2'-fluoro modifications of the ribose sugar were incorporated at specific positions within SCAP siRNA. Phosphorothioate internucleotide linkages were also incorporated at the ends of the antisense and / or sense sequences. Table 2 below shows the modifications in the sense and antisense sequences for each modified SCAP siRNA. The nucleotide sequences in Table 2 are listed according to the following notation: A, U, G, and C = corresponding ribonucleotides; dc and dG = corresponding deoxyribonucleotides; dT = deoxythymidine; a, u, g, and c = corresponding 2'-O-methylribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; [InvAb] is an inverted abasic residue; [Ab] is an abasic residue; GNA is glycol nucleic acid, and bases with a GNA backbone are designated AgN, UgN, CgN, and GgN. The insertion of an "s" in a sequence indicates that two adjacent nucleotides are linked by a phosphorothioate group (e.g., a phosphorothioate internucleotide linkage). Unless otherwise indicated, all other nucleotides are linked by a 3'-5' phosphodiester group. Each of the siRNA compounds in Table 2 contains a 19-21 base pair duplex region with a 2-nucleotide overhang at the 3' end of both strands or a blunt end at one or both ends. Each [phosphate] was attached to the following GalNAc structure: [ka] wherein X=O or S.

[0183] [Table 6]

[0184] [Table 7]

[0185] [Table 8]

[0186] [Table 9]

[0187] [Table 10]

[0188] [Table 11]

[0189] [Table 12]

[0190] [Table 13]

[0191] Example 2: Efficacy of selected SCAP siRNA molecules in an RNA FISH assay A panel of fully chemically modified siRNAs was prepared and tested in vitro for mRNA knockdown potency and selectivity. Each siRNA duplex consisted of two strands: a sense or "passenger" strand and an antisense or "guide" strand. The substitution of the native 2'-OH on the ribose of specific nucleotides was described in Example 1. In some cases, the phosphodiester internucleotide linkage in one or both strands was replaced with phosphorothioate to reduce exonucleolytic degradation.

[0192] RNA FISH (fluorescence in situ hybridization) assays were performed to measure SCAP mRNA knockdown by test siRNA. Hep3B cells (purchased from ATCC) were cultured in minimum essential medium (MEM, Corning) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Corning). siRNA transfection was performed as follows: 1 μL of test siRNA and 4 μL of plain MEM were added to a PDL-coated CellCarrier-384 Ultra assay plate (PerkinElmer) using a BioMek FX (Beckman Coulter). Next, 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) prediluted in plain MEM (0.035 μL of RNAiMAX in 5 μL MEM) was dispensed into the assay plate using a Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific). After a 20-minute incubation of the siRNA / RNAiMAX mixture at room temperature (RT), 30 μL of Hep3B cells (2,000 cells per well) in MEM supplemented with 10% FBS and 1% PS was added to the transfection complex using a Multidrop Combi Reagent Dispenser. The assay plate was allowed to settle for 20 minutes at room temperature before being moved to an incubator. Cells were incubated at 37°C and 5% CO for 72 hours. The ViewRNA ISH cell assay was performed according to the manufacturer's protocol (Thermo Fisher Scientific) using an in-house assembled automated FISH assay platform for liquid handling. Briefly, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 minutes at RT, permeabilized with detergent for 3 minutes at RT, and then treated with protease solution for 10 minutes at RT.Incubation of the target-specific probe pair (Thermo Fisher Scientific) was performed for 3 hours, while incubation of the preamplifier, amplifier, and label probe (Thermo Fisher Scientific) was performed for 1 hour each. All hybridization steps were performed at 40°C in a Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific). After the hybridization reaction, cells were stained with Hoechst and CellMask Blue (Thermo Fisher Scientific) for 30 minutes and then imaged using an Opera Phenix (PerkinElmer). Images were analyzed using a Columbus Image Data Storage and Analysis System (PerkinElmer) to obtain the average spot count per cell. Spot counts were normalized using high (containing phosphate-buffered saline, Corning) and low (no target probe pair) control wells. Normalized values ​​were plotted against total siRNA concentration, and data were fitted to a four-parameter sigmoidal model using Genedata Screener (Genedata) to obtain IC50 and maximal activity.

[0193] The results of the RNA FISH assay for Hep3B cells are shown in Table 3. Values ​​represent knockdown of SCAP mRNA.

[0194] [Table 14]

[0195] Example 3: In vivo silencing studies testing the silencing efficacy of SCAP siRNA sequences Nine- to ten-week-old C57B16 males were obtained from Charles River Laboratories and housed in accordance with Amgen guidelines and Institutional Animal Care and Use Committees (IACUC) protocols. The animals were randomized according to body weight, with six animals randomly assigned to each siRNA trigger sequence. On day 0, cohorts received a single subcutaneous dose of PBS or a specific siRNA compound at 3 mg / kg body weight. On day 29, mice were euthanized under CO2, and the left liver lobe was harvested from each animal. The tissue was cut into small pieces and immediately snap-frozen in liquid nitrogen for further downstream assays.

[0196] RNA was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer's guidelines. 2 μg of RNA was treated with DNase I (Promega) and subjected to quantitative PCR using the Taqman RNA to CT, One Step Kit (Thermo Fisher Scientific). mRNA expression was quantified using gene-specific Taqman probes for mouse SCAP and GAPDH. GAPDH was used as an internal control. qPCR experiments were performed using an Invitrogen QuantStudio7 Flex Real-Time PCR System. Expression levels were calculated using the delta CT method.

[0197] In vivo screening with 9-10 week old Ob / Ob mice (Ob / Ob on a Bl6 background) from Jackson Laboratories was performed using the same method as described for C57Bl6 mice.

[0198] All animal experiments described herein were approved by Amgen's Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, 8 thMice were cared for in accordance with the National Research Council (US) Guide for the Care and Use of Laboratory Animals., Institute for Laboratory Animal Research (US) and National Academies Press (US) (2011) Guide for the care and use of laboratory animals. 8th Ed., National Academies Press, Washington, DC. Mice were kept in an air-conditioned room for 22 ± 2 ℃ Animals were housed singly under a 12-hour light:12-hour dark cycle (0600-1800 h). Animals had ad libitum access to regular chow diet (Envigo, 2920X) and water (reverse osmosis purified) via an automated watering system unless otherwise indicated. At termination, blood was collected by cardiac puncture under deep anesthesia before euthanasia by secondary physical methods in accordance with Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.

[0199] Data on relative knockdown are shown in Table 4, which shows the relative knockdown at a dose of 3 mg / kg on day 25. SCAP knockdown is the percentage reduction in SCAP mRNA levels.

[0200] [Table 15]

[0201] Example 4: Efficacy of selected SCAP siRNA molecules in an RNA FISH assay A panel of fully chemically modified siRNAs was prepared and tested in vitro for mRNA knockdown potency and selectivity. Each siRNA duplex consisted of two strands: a sense or "passenger" strand and an antisense or "guide" strand. The substitution of the native 2'-OH on the ribose of specific nucleotides was described in Example 1. In some cases, the phosphodiester internucleotide linkage in one or both strands was replaced with phosphorothioate to reduce exonucleolytic degradation.

[0202] RNA FISH was performed as described in Example 2. Cells were incubated at 37°C and 5% CO2 for 72 hours. The ViewRNA ISH cell assay was performed according to the manufacturer's protocol (Thermo Fisher Scientific) using an in-house assembled automated FISH assay platform for liquid handling. Briefly, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 minutes at room temperature, permeabilized with detergent for 3 minutes at room temperature, and then treated with protease solution for 10 minutes at room temperature. Incubation with the target-specific probe pair (Thermo Fisher Scientific) was performed for 3 hours, while incubation with the preamplifier, amplifier, and label probe (Thermo Fisher Scientific) was performed for 1 hour each. All hybridization steps were performed at 40°C in a Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific). After the hybridization reaction, cells were stained with Hoechst and CellMask Blue (Thermo Fisher Scientific) for 30 minutes and then imaged using Opera Phenix (PerkinElmer). Images were analyzed using a Columbus Image Data Storage and Analysis System (PerkinElmer) to obtain the average number of spots per cell. Spot counts were normalized using high (containing phosphate-buffered saline, Corning) and low (no target probe pair) control wells. Normalized values ​​were plotted against total siRNA concentration, and the data were fitted to a four-parameter sigmoidal model using Genedata Screener (Genedata) to obtain IC50 and maximum activity.

[0203] The results of the RNA FISH assay on Hep3B cells are shown in Table 5 for duplexes D-2080 to D-2109, Table 6 for triggers D-2110 to D-2124, and Table 7 for triggers D-2125 to D2146. Negative values ​​for maximum activity indicate knockdown of activity.

[0204] [Table 16]

[0205] [Table 17]

[0206] [Table 18]

[0207] Example 5: Screening of siRNA triggers modified with destabilizing bases Nine- to ten-week-old C57Bl6 / J male mice were obtained from Charles River Laboratories and acclimated in-house. Mice were weighed and randomized into groups of eight animals. These animals were subcutaneously dosed with SCAP siRNA trigger at 3 mg / kg body weight. Stock siRNA compounds were diluted in calcium- and magnesium-free phosphate buffer solution (Thermo Fischer Scientific, 14190-136) immediately prior to dosing. Thirty days after siRNA treatment, animals were euthanized and livers were harvested. Freshly isolated left liver lobes were immediately snap-frozen in liquid nitrogen. RNA was isolated from 30 to 50 mg of liver tissue using the QIAcube HT instrument RNeasy 96 QIAcube HT kit according to the manufacturer's protocol. 2 to 4 μg of RNA was treated with RQ1 RNase-Free DNase (Promega, M6101). Ten nanograms of DNase-digested RNA was subjected to real-time qPCR using the TaqMan RNA to CT 1-step kit (Applied Biosystems) run on a Quant Studio Real Time PCR machine. TaqMan probes for mouse SCAP (Mm01250176_m1) and GAPDH (4352932E) were used to calculate the fold change in SCAP expression in the SCAP siRNA-treated group compared to the PBS (buffer control) group. Data are expressed as percent knockdown in the siRNA-treated group relative to the PBS group. Five trigger sequences with various destabilizing modifications were tested: D-2040, D-2041, D-2042, D-2044, and D-2045. Destabilizing base modifications included both GNA and abasic modification patterns. The data are shown in Table 8. In each case, modification patterns containing destabilizing bases resulted in lower SCAP mRNA expression compared to the parent trigger modification. Duplexes are shown as % SCAP knockdown.

[0208] [Table 19]

[0209] Example 6: Screening of siRNA triggers in C57B16 / J male mice The D-2040, D-2045, D-2042, D-2041, and D-2044 sequences were additionally modified with different chemical modification patterns. These modified triggers were compared with the original trigger modification patterns. Groups 1–5 contain trigger sequence D-2040 and variations in the modification patterns. Groups 6–10 contain trigger sequence D-2045 and variations in the modification patterns. Groups 11–15 contain trigger sequence D-2042 and variations in the modification patterns. Groups 16–21 contain trigger sequence D-2041 and variations in the modification patterns. Groups 22–27 contain trigger sequence D-2044 and variations in the modification patterns. For the survival phase of the in vivo study, 13–15-week-old C57B16 / J male mice were obtained from Charles River Laboratories and acclimatized in-house. Mice were weighed and randomized into groups of eight animals each. These animals were subcutaneously dosed with SCAP siRNA trigger at 3 mg per kg body weight. Stock siRNA compounds were diluted in calcium- and magnesium-free phosphate buffer solution (Thermo Fischer Scientific, 14190-136) prior to dosing. Thirty days after siRNA treatment, animals were euthanized and livers were harvested. Freshly isolated left liver lobes were immediately flash-frozen in liquid nitrogen. RNA was isolated from 30–50 mg of liver tissue using the RNeasy 96 QIAcube HT kit on a QIAcube HT instrument according to the manufacturer's protocol. 2–4 μg of RNA was treated with RQ1 RNase-Free DNase (Promega, M6101). 10 ng of DNase-digested RNA was subjected to real-time qPCR using the TaqMan RNA to CT 1-step kit (Applied Biosystems) run on a Quant Studio Real Time PCR machine. TaqMan probes for mouse SCAP (Mm01250176_m1) and GAPDH (4352932E) were used to calculate the fold change in SCAP expression in the siRNA-treated groups compared with the PBS (buffer control) group. The data are shown in Table 9 and are expressed as percent knockdown in the siRNA-treated groups relative to the PBS group.As shown, different modification patterns result in different levels of silencing.

[0210] [Table 20]

[0211] Example 7: siRNA-triggered silencing of SCAP in Ob / Ob animals Ten- to twelve-week-old male B6.V-Lep ob / J (632) mice (also known as Ob / Ob mice) were obtained from Jackson Laboratories. After acclimation, the animals were randomized into groups of n = 8. Mice were treated with chemically modified SCAP siRNA triggers identified from the screening experiments in the previous example. Mice were subcutaneously dosed with 3 milligrams of siRNA per kilogram of body weight. Animals were sacrificed 20 and 30 days after siRNA administration. After euthanasia, the left liver lobe was isolated and immediately snap-frozen in liquid nitrogen. RNA was isolated from 30 to 50 mg of liver tissue using the QIAcube HT instrument RNeasy 96 QIAcube HT kit according to the manufacturer's protocol. 2 to 4 μg of RNA was treated with RQ1 RNase-Free DNase (Promega, M6101). Ten nanograms of DNase-digested RNA was subjected to real-time qPCR using the TaqMan RNA to CT 1-step kit (Applied Biosystems) run on a Quant Studio Real Time PCR machine. TaqMan probes for mouse SCAP (Mm01250176_m1) and GAPDH (4352932E) were used to calculate the fold change in SCAP expression in the siRNA-treated group compared to the PBS (buffer control) group. The data are shown in Table 10 and are expressed as percent knockdown in the siRNA-treated group relative to the PBS group.

[0212] [Table 21]

[0213] Example 8: Efficacy Study with SCAP Trigger Prevention and rescue of NASH phenotypes in efficacy models 1] Amylin (AMLN) AMLYN model An amylin liver NASH (AMLN) model was developed by feeding 5-week-old obese male mice (Ob / Ob) obtained from Jackson Laboratories, strain B6.V-Lep ob / J(632), with a high-fat, high-cholesterol diet. The 45% fat, 36% carbohydrate, and 2% cholesterol diet was obtained from Envigo, catalog number TD170748. Regular water was replaced with a sugar solution containing 55% fructose and 46% glucose in water. Mice were randomized into eight groups and treated with Trigger D-2040, Trigger D-2147, or PBS. D-2147 is a seed sequence-matched control for Trigger D-2040 in which nucleotides 9-11 are switched. Mice were subcutaneously dosed with the AMLN diet at 3 mg / kg body weight Q2D at weeks 8, 10, and 12. Stock siRNA compounds were diluted in calcium- and magnesium-free phosphate buffer solution (Thermo Fischer Scientific, 14190-136) immediately before dosing. After 6 weeks of continuous SCAP silencing, mice were harvested at week 14 of diet. During harvest, after euthanasia under isoflurane, the liver median lobe was fixed in 10% neutral buffered formalin. Formalin-fixed median lobes were further processed for immunohistochemistry (IHC) using hematoxylin and eosin (Dako, CS70030-2, CS70130-2), trichrome staining, and alpha-smooth muscle actin (aSMA) expression according to the manufacturer's instructions. NASH readout was performed by scoring for fibrosis and stellate cell activation, and readings were performed by a board-certified pathologist.

[0214] The left liver lobe was snap-frozen in liquid nitrogen. The snap-frozen tissue was further processed for RNA extraction and gene expression assessment as detailed in Example 7. Additionally, liver triglyceride content was measured by homogenizing 50-100 mg of snap-frozen liver tissue in isopropanol. Samples were homogenized and incubated on ice for 1 hour, then spun at 10,000 rpm for 10 minutes. The supernatant was transferred to a clean 96-deep well plate. Triglyceride content was determined by a colorimetric assay (Infinity Triglyceride Reagent, Thermo Fisher Scientific, TR22421) and using a standard (Pointe Scientific, T7531-STD) according to the manufacturer's instructions. Data are expressed as milligrams of triglyceride per milligram of tissue.

[0215] Additional endpoints captured during harvest included measuring liver weight. The ratio of total liver weight (grams) to end-stage body weight (grams) was analyzed to monitor changes in liver mass. SCAP silencing inhibits PCSK9 expression. Serum PCSK9 levels were measured as a biomarker using an ELISA assay (R&D Systems, MPC900).

[0216] Figure 1A shows SCAP mRNA expression expressed as fold change relative to the PBS control group. The Trigger D-2040-treated group achieved approximately 85% SCAP silencing (85.3%), while there was no significant change in the D-2147-treated group. Figure 1B shows a significant reduction in end-stage liver weight:body weight ratio in Trigger D-2040-treated mice. Figure 1C shows a reduction in liver triglycerides in Trigger D-2040-treated mice, while they remained unchanged in the D-2147-treated group. In Figure 1D, serum PCSK9 levels were measured. Effective SCAP silencing significantly reduced serum PCSK9 levels. Figures 1E and 1F show pathological readouts of fibrosis (trichrome staining) and stellate cell activation (aSMA immunohistochemistry). SCAP silencing with Trigger D-2040 significantly reduced fibrosis scores, suggesting improved NASH outcomes. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison test. Asterisks indicate adjusted p-values ​​(****p-value<0.0001, ***p-value<0.001).

[0217] 2] ALIOS model We also tested the efficacy of SCAP triggers in the American Lifestyle Induced Obesity Syndrome mouse model (ALIOS). C57B16 male mice obtained from Charles River Laboratories at 5 weeks of age were fed the ALIOS diet, which is similar to the AMLN diet except for its reduced cholesterol content. The ALIOS diet contained 0.2% cholesterol and was obtained from Envigo (catalog number TD130885). Regular water was replaced with a sugar solution containing 55% fructose and 46% glucose in water. After 18 weeks of feeding the animals with the ALIOS diet, the mice were subcutaneously dosed with 3 milligrams per kilogram of body weight every other week for 6 weeks. Mice were randomized into groups of 4-5 and treated with Trigger D-2040, Trigger D-2042, or PBS. Mice were harvested after 6 months of diet and 6 weeks of SCAP silencing. Similar to previous efficacy studies, endpoint analyses included SCAP message levels, end-stage liver-to-body weight ratio, liver triglyceride levels, serum PCSK9 levels, and pathological readouts of fibrosis using trichrome staining and stellate cell activation using alpha-smooth muscle actin immunohistochemical staining.

[0218] Figure 2A shows SCAP mRNA expression. Data are expressed as fold change relative to the PBS group. Both SCAP triggers D-2040 and D-2042 showed a greater than 85% reduction in SCAP mRNA. In Figure 2B, a significant reduction was observed in the end-stage liver weight / body weight (LW / BW) ratio in the groups treated with the SCAP triggers D-2040 and D-2042. Figure 2C shows liver triglyceride levels in various groups. Administration of SCAP triggers D-2040 and D-2042 significantly reduced liver triglyceride content compared with the buffer control group. In Figure 2D, serum PCSK9 was measured using the ELISA kit described previously. Serum PCSK9 levels in the SCAP siRNA-treated group were significantly reduced compared with the PBS group. Figures 2E and 2F show pathological readouts of fibrosis measured by Trichrome staining and immunostaining for alpha-smooth muscle actin, indicating stellate cell activation. Both readouts show a reduction after SCAP silencing, suggesting rescue of the NASH phenotype after SCAP silencing. Statistical significance was determined by one-way ANOVA with Dunnett's multiple comparison test. Asterisks indicate adjusted p-values ​​(****p<0.0001, ***p<0.005, **p<0.01, and *p<0.05).

[0219] Example 9: Use of the DIAMOND model to test the efficacy of SCAP siRNA to treat hepatocellular carcinoma More than 50% of hepatocellular carcinoma patients suffer from nonalcoholic fatty liver disease. To test the efficacy of SCAP siRNA in preventing further HCC progression, we developed a model in which HCC is manifested by a long-term NASH diet without chemical modifiers. Such a model more fully represents human pathophysiology. One such model is the diet-induced animal model of nonalcoholic fatty liver disease (or DIAMOND). It was developed using a unique syngeneic animal strain derived from the C57Bl / 6J and 1291SvImJ backgrounds. Starting at 8 weeks of age, male mice from this intergenic colony are fed a high-fat, high-carbohydrate diet (42% kcal from fat) containing 0.1% cholesterol. Drinking water is also replaced with a high-fructose-glucose solution. After 32 weeks on this diet, the model develops HCC. By 51 weeks, DIAMOND model liver tissue displays large areas of tumors and foci of alterations within hepatocytes. The model is also highly penetrant. To test efficacy, SCAP siRNA and vehicle control are administered at week 40 of the diet. Mice are re-administered with vehicle or SCAP siRNA at regular intervals for 6–10 weeks to ensure downregulation of SCAP gene expression. Endpoint analyses include pathological examination of hepatocellular tumor burden metastatic tumor index, assessment of tumor growth using Ki67 expression, and the degree of tumor angiogenesis using CD31 expression. qPCR and protein analysis are also performed to confirm effective silencing of targets and downstream pathways.

[0220] Example 10: Evaluation of SCAP siRNA in the Huh-7 liver xenograft model of HCC SCAP siRNA was evaluated using an orthotopic Huh-7 liver xenograft model. Six-week-old BALB / c athymic nude mice were intrahepatically injected with one million Huh-7 cells suspended in cell culture medium containing 33% Matrigel. Mice were then divided into groups for treatment with vehicle or SCAP siRNA. Vehicle or SCAP siRNA was re-administered at regular intervals (e.g., every other week) to ensure sustained reduction of SCAP mRNA. At various time points (e.g., 4 weeks) after the initial vehicle or siRNA treatment, mice were euthanized, and livers were harvested and fixed in 4% paraformaldehyde. To understand the efficacy of SCAP siRNA treatment, tumor burden was measured. qPCR and protein analysis were also evaluated to confirm effective target silencing.

Claims

1. 1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense sequence listed in Table 1 or Table 2, and wherein the RNAi construct inhibits expression of SREBP cleavage activating protein (SCAP).

2. The RNAi construct of claim 1 , wherein the antisense strand comprises a region complementary to an mRNA sequence of SCAP.

3. 3. The RNAi construct of claim 1 or 2, wherein the sense strand comprises a region having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense sequence listed in Table 1 or Table 2.

4. The RNAi construct of any one of claims 1 to 3, wherein the sense strand comprises a sequence that is sufficiently complementary to a sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length.

5. The RNAi construct of claim 4, wherein the duplex region is about 17 to about 24 base pairs in length.

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

7. The RNAi construct of claim 6 , wherein the duplex region is 19 base pairs in length.

8. The RNAi construct according to any one of claims 4 to 7, wherein the sense strand and the antisense strand are each about 15 to about 30 nucleotides in length.

9. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each about 19 to about 27 nucleotides in length.

10. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each about 21 to about 25 nucleotides in length.

11. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each about 21 to about 23 nucleotides in length.

12. The RNAi construct of any one of claims 1 to 11, comprising at least one blunt end.

13. 12. The RNAi construct of any one of claims 1 to 11, comprising at least one nucleotide overhang of 1 to 4 unpaired nucleotides.

14. The RNAi construct of claim 13 , wherein the nucleotide overhang has two unpaired nucleotides.

15. 15. The RNAi construct of claim 13 or 14, comprising a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' end of both the sense strand and the antisense strand.

16. The RNAi construct of any one of claims 13 to 15, wherein the nucleotide overhang comprises a 5'-UU-3' dinucleotide or a 5'-dTdT-3' dinucleotide.

17. The RNAi construct of any one of claims 1 to 16, comprising at least one modified nucleotide.

18. The RNAi construct of claim 17, wherein the modified nucleotide is a 2'-modified nucleotide.

19. 18. The RNAi construct of claim 17, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a glycol nucleic acid, an inverted base, or a combination thereof.

20. 20. The RNAi construct of claim 19, wherein the modified nucleotides are 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

21. 18. The RNAi construct of claim 17, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides.

22. 22. The RNAi construct of claim 21, wherein the modified nucleotides are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

23. 23. The RNAi construct of any one of claims 1 to 22, comprising at least one phosphorothioate internucleotide linkage.

24. 24. The RNAi construct of claim 23, comprising two consecutive phosphorothioate internucleotide linkages at the 3' end of the antisense strand.

25. The RNAi construct of claim 23, wherein the antisense strand contains two consecutive phosphorothioate internucleotide bonds at both the 3'-end and 5'-end, and the sense strand contains two consecutive phosphorothioate internucleotide bonds at the 5'-end.

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

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

28. 28. The RNAi construct of any one of claims 1 to 27, which is any one of the double-stranded compounds listed in any one of Tables 1 to 2.

29. 29. The RNAi construct of any one of claims 1 to 28, which reduces the expression level of SCAP in hepatocytes after incubation with the RNAi construct compared to the expression level of SCAP in hepatocytes incubated with a control RNAi construct.

30. 30. The RNAi construct of claim 29, wherein the hepatocyte is a Hep3B cell.

31. 31. The RNAi construct of any one of claims 1 to 30, which inhibits at least 10% of SCAP expression in Hep3B cells in vitro at 5 μM.

32. 31. The RNAi construct of any one of claims 1 to 30, which inhibits SCAP expression in Hep3B cells with an IC50 of less than about 1 nM.

33. A pharmaceutical composition comprising the RNAi construct of any one of claims 1 to 32 and a pharmaceutically acceptable carrier, excipient, or diluent.

34. A method for reducing the expression of SCAP in a patient in need thereof, comprising administering to said patient an RNAi construct according to any one of claims 1 to 32.

35. The method of claim 34, wherein the expression level of SCAP in hepatocytes is reduced in the patient after administration of the RNAi construct compared to the expression level of SCAP in a patient who has not been administered the RNAi construct.

36. A method for treating a subject suffering from a SCAP-associated disease, comprising administering to the subject an RNAi construct described in any one of claims 1 to 32 or a pharmaceutical composition of claim 33.

37. The method of claim 36, wherein administering to the subject an RNAi construct of any one of claims 1 to 32 or a pharmaceutical composition of claim 33 results in a reduction in expression of the SCAP gene by at least about 10%.

38. 37. The method of claim 36, wherein the disease is selected from the group consisting of fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia, or non-alcoholic fatty liver disease (NAFLD).

39. 34. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense sequence listed in Table 1 or Table 2, and used in a method for treating a SCAP-associated disease, the method comprising administering to the subject the RNAi construct of any one of claims 1 to 32 or the pharmaceutical composition of claim 33.

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