RNAI constructs for inhibiting SCAP expression and methods of using the same

Abstract

The present disclosure relates to RNAi constructs, such as siRNA, for reducing the expression of the SCAP gene. Methods of using such RNAi constructs for treating or preventing liver diseases such as non-alcoholic fatty liver disease (NAFLD) are also described. In certain embodiments, the RNAi construct comprises a region having at least 15 contiguous nucleotides that differ from the antisense sequences listed in Table 1 by 3 nucleotides or less.

Description

Technical Field

[0001] The present disclosure relates to compositions and methods for modulating the hepatic expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP). In particular, the present disclosure relates to nucleic acid-based therapeutics for reducing SCAP expression via RNA interference and methods of treating or preventing liver diseases, such as non-alcoholic fatty liver disease (NAFLD), using such nucleic acid-based therapeutics.

[0002] Incorporation by reference of electronically submitted materials The following nucleotide / amino acid sequence listing, identified as follows and filed simultaneously with this specification, is hereby incorporated by reference in its entirety: one XML document of 550 kilobytes named "A-2911-WO01-SEC.xml" created on May 19, 2023.

[0003] Cross-reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 345,513, filed May 25, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0004] Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, including a variety of liver pathologies. Its prevalence has doubled in the past 20 years and is currently estimated to affect approximately 20% of the world's 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 significant history of alcohol consumption and 2) have been excluded from the diagnosis of other types of liver diseases (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, induces inflammation and hepatocyte injury, leading to a more advanced stage of the disease called non-alcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, it was predicted that 75 million to 100 million Americans had NAFLD, and NASH accounts for approximately 10-30% of NAFLD diagnoses (Rinella, supra; Younossi et al (2016) Hepatology 64(5):1577-1586).

[0005] One of the important triggers for the development of NAFLD is the loss of triglyceride (TG) homeostasis in the liver, which leads to an increase in the accumulation of TG and fatty acids. Over time, lipotoxicity exhausts the adaptive and regenerative responses of hepatocytes, conferring inflammation, activation of the innate immune system, and steatohepatitis. Progressive NASH causes collagen accumulation and fibrosis in the liver. As the grade of fibrosis worsens, 10% - 29% of the livers of NASH patients develop cirrhosis and ultimately lead to hepatocellular carcinoma.

[0006] The sterol regulatory element-binding protein (SREBP) family plays an important role in the regulation of de novo lipogenesis and TG accumulation in the liver. SREBP is synthesized as an inactive precursor in the endoplasmic reticulum (ER). The SREBP cleavage-activating protein (SCAP) is the only known regulator of the transcription factors of the SREBP family. Immediately after synthesis, SCAP forms a complex with SREBP and escorts SREBP to the Golgi vesicles. Here, SREBP is further processed to release the active amino terminus of the transcription factor. The active SREBP translocates to the nucleus, binds to the SREBP response element, and drives the transcriptional activation of target genes. Therefore, inhibition of SCAP function can prevent the processing of active SREBP and reduce lipogenesis and TG accumulation in the liver. Currently, since there is no approved pharmacological treatment for NAFLD symptoms, it is managed by weight loss and treatment of any secondary symptoms. Therefore, there is a need for compositions and methods for treating NAFLD in affected individuals.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0008] The present disclosure provides an RNAi construct comprising a sense strand and an antisense strand, which inhibits the expression of the mRNA of sterol regulatory element-binding protein cleavage-activating protein (SCAP). In certain embodiments, the RNAi construct comprises a region having at least 15 contiguous nucleotides that differ from the antisense sequences listed in Table 1 by 3 nucleotides or less.

[0009] In some embodiments, the sense strand of the RNAi construct described herein comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a double-stranded region that is 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 nucleotide overhangs 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 an overhang of two unpaired nucleotides at the 3' ends of the sense and antisense strands. In other embodiments, the RNAi construct comprises an overhang of two unpaired nucleotides at the 3' end of the antisense strand and a blunt end at the 3' end of the sense strand / 5' end of the antisense strand.

[0010] The RNAi constructs of the present disclosure may include one or more modified nucleotides, including nucleotides having modifications to the ribose ring, nucleobase, or phosphodiester backbone. In some embodiments, the modification to the ribose ring of the RNAi construct includes one or more 2'-modifications. Such 2'-modifications can include 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-allyl-modified nucleotides, or bicyclic nucleic acids (BNA). The modification to the ribose ring may include the incorporation of glycol nucleic acid (GNA), where the ribose ring is replaced by propylene glycol. In a particular embodiment, the RNAi construct includes one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or a combination thereof. In some embodiments, all nucleotides of the sense and antisense strands of the RNAi construct are modified nucleotides. The RNAi constructs of the present disclosure may incorporate abasic nucleotides as terminal nucleotides, for example, at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand. In such embodiments, the abasic nucleotides can be inverted and can be linked to adjacent nucleotides via, for example, 3'-3' nucleotide linkages or 5'-5' nucleotide linkages.

[0011] In some embodiments, the RNAi construct includes at least one backbone modification, such as a modified nucleotide linkage or a nucleoside linkage. In certain embodiments, the RNAi constructs described herein include at least one phosphorothioate nucleotide linkage. In a particular embodiment, the phosphorothioate nucleotide linkage may be located at the 3'-end or 5'-end of the sense strand and / or the antisense strand.

[0012] The present disclosure also provides a composition comprising the aforementioned RNAi construct and a pharmaceutically acceptable carrier, excipient, or diluent, and a method of reducing the expression of SCAP in a patient in need thereof, the method comprising administering the aforementioned RNAi construct or composition to the patient.

[0013] The present disclosure also includes an RNAi construct for use in reducing the expression of SCAP, such as for the treatment of NAFLD or NASH. Also provided is the use of an RNAi construct that inhibits SCAP expression for the preparation of a medicament for treating NAFLD.

BRIEF DESCRIPTION OF THE INVENTION

[0014] The present disclosure is based in part on the design and generation of an RNAi construct that targets the sterol regulatory element-binding protein cleavage-activating protein (SCAP) gene and reduces the expression of SCAP in liver cells. Inhibition of SCAP expression is useful for treating or preventing conditions associated with SCAP expression, such as simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible advanced scarring of the liver), or SCAP-mediated hyperlipidemia or hypertriglyceridemia.

[0015] The present disclosure provides compositions and methods for regulating the expression of genes encoding SCAP. In some embodiments, the gene can be present within a cell or a subject such as a mammal (e.g., a human). In some embodiments, the compositions of the present disclosure include RNAi constructs that target SCAP mRNA and reduce SCAP expression in a cell or a mammal. Such RNAi constructs are useful for treating or preventing various forms of liver-related diseases such as simple fatty liver (steatosis), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible advanced scarring of the liver), or SCAP-mediated hyperlipidemia or hypertriglyceridemia (see, for example, Lee et al., Experimental & Molecular Medicine, 52:724-729 (2020)).

[0016] As described above, SCAP (SREBP cleavage-activating protein) is a sterol regulatory escort protein that transports SREBPs from their sites of synthesis in the endoplasmic reticulum to their cleavage sites in the Golgi apparatus, where they are sequentially cleaved by two proteases that release the cytoplasmic NH 2 terminal transcription factor domain. Cell culture experiments have demonstrated that mutant cells lacking SCAP have low levels of SREBP precursors, which is apparently because these proteins are unstable in the absence of SCAP. In addition, SCAP-deficient CHO cells are unable to synthesize cholesterol and they require an external source of cholesterol for growth (Rawson et al., J Biol Chem., 274:28549-28556 (1999)). SCAP also functions as a cholesterol sensor, which is mediated by the multiple transmembrane domains of SCAP. Point mutations in this sterol-sensing motif prevent sterol inhibition of SREBP cleavage and result in uncontrolled overproduction of cholesterol.

[0017] The control of lipid synthesis is particularly important in the liver, which synthesizes lipids not only for its own use but also for transfer into the plasma as lipoproteins. The level of plasma lipoprotein cholesterol is decreased by treatment with statins. In addition, hepatic fatty acid synthesis increases when plasma insulin rises, as can occur in obesity and non-insulin-dependent type II diabetes. In the liver, overexpression of the soluble transcriptional activation domain of SREBP-1a in transgenic mice results in massive fatty liver. Transgenic mice overexpressing the sterol-resistant mutant SCAP produce excessive cholesterol and give rise to livers that do not exhibit normal feedback inhibition when cholesterol is ingested, suggesting that SCAP plays a regulatory role in the liver. Mice with conditional SCAP deficiency in the liver show a reduction in the basal rates of cholesterol and fatty acid synthesis in the liver, mainly due to a decrease in the mRNAs encoding multiple biosynthetic enzymes (Matsuda et al., Genes & Development 15:1206-121(2001). In addition, patients with NASH / NAFLD show increased expression and transcriptional activity of SREBP1c and its target genes. Collectively, these data provide in vivo evidence that SCAP and SREBP are required for hepatic lipid synthesis.

[0018] RNA interference (RNAi) is a process in which foreign RNA is introduced into cells, resulting in specific degradation of the mRNA encoding the target protein and, as a result, reduced protein expression. Advances in both RNAi technology and delivery to the liver, along with the favorable outcomes increasing with other RNAi-based therapies, suggest that directly targeting genes that regulate lipid synthesis in the liver, such as SCAP, is a promising means for therapeutically treating NAFLD.

[0019] As used herein, the term "RNAi construct" refers to an agent that includes an RNA molecule that, when introduced into a cell, can down-regulate 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, the RNAi construct includes a double-stranded RNA (dsRNA) molecule that comprises two antiparallel strands of contiguous nucleotides that are sufficiently complementary to each other to hybridize and form a double-stranded region. The double-stranded RNAi construct may also be referred to as an RNAi "trigger". The term "hybridize" or "hybridization" typically refers to the pairing of complementary polynucleotides via hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. A strand that includes a region having a sequence that is substantially complementary to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand". The "sense strand" refers to a strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may include a region having a sequence that is substantially identical to the target sequence.

[0020] In certain embodiments, the sense and antisense strands of the double-stranded RNA hybridize to form a double-stranded region, but otherwise can be two separate molecules that are separated. Such double-stranded RNA molecules formed from two separate strands are referred to as "small interfering RNA" or "short interfering RNA" (siRNA). siRNA is typically about 20-27 base pairs and is a class of non-coding double-stranded RNA molecules that are central to RNAi. Thus, in some embodiments, the RNAi constructs of the present disclosure include siRNA. In other embodiments, the RNAi construct can be a microRNA (also known as "miRNA" or "mature miRNA"). miRNA are small (approximately 18-24 nucleotides in length) non-coding RNA molecules that are present in plants, animals, and some viruses. miRNA are similar to siRNA, but miRNA are derived from endogenous precursor hairpin RNA structures. miRNA regulate gene expression by leading to base pairing to complementary regions of the target mRNA and cleavage of the target RNA via the RISC pathway.

[0021] In some embodiments, the present disclosure provides an RNAi construct directed to SCAP. In some embodiments, the RNAi construct is an siRNA comprising a sense strand and an antisense strand, and the antisense strand comprises a region that is complementary to a region of the mRNA sequence of SCAP. The region of the RNAi antisense strand can be complementary to any suitable region of the mRNA sequence of SCAP. For example, the antisense strand can comprise a region that is complementary to the coding region or the 3' untranslated region (UTR) of the mRNA sequence of SCAP.

[0022] The double-stranded RNAi molecule can include chemical modifications to the ribonucleotides, including modifications to the ribose sugar, base, or backbone components such as those described herein or known in the art. Any such modifications as used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term "double-stranded RNA" for the purposes of the present disclosure.

[0023] As used herein, a polynucleotide containing a first sequence is "complementary" to a polynucleotide containing a second sequence if, under certain conditions such as physiological conditions, the polynucleotide containing the first sequence can hybridize to the polynucleotide containing the second sequence to form a double-stranded region. Other such conditions can include moderate or stringent hybridization conditions known to those skilled in the art. A first sequence is considered to be "perfectly complementary" (100% complementary) to a second sequence if the polynucleotide containing the first sequence base pairs with the polynucleotide containing the second sequence without any mismatches over the entire length of one or both nucleotide sequences. A sequence is "substantially complementary" to a target sequence if it is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the target sequence. The percentage of complementarity can be calculated by dividing the number of bases in the first sequence that are complementary to the bases at the corresponding positions in the second or target sequence by the total length of the first sequence. Also, if, when two sequences hybridize, there are 5, 4, 3, 2, or 1 or fewer mismatches over the entire 30-base pair double-stranded region, one sequence may be said to be substantially complementary to the other sequence. Generally, in the presence of any nucleotide overhang as defined herein, the sequence of such an overhang is not considered in determining the degree of complementarity between the two sequences. By way of example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand that hybridize to form a 19-base pair double-stranded region with a 2-nucleotide overhang at the 3' end of each strand would be considered to be perfectly complementary when this term is used herein.

[0024] In some embodiments, the region of the antisense strand comprises a sequence that is perfectly 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 perfectly 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 having 1, 2, 3, 4, or 5 mismatches within the double-stranded region formed by the sense and antisense strands. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g., within 6, 5, 4, 3, 2, or 1 nucleotide of the 5' and / or 3' ends of the strand). In one embodiment, any mismatches within the double-stranded region formed by the sense and antisense strands preferably occur within 6, 5, 4, 3, 2, or 1 nucleotide of the 5' end of the antisense strand.

[0025] When the two substantially complementary strands of the dsRNA are constituted by separate RNA molecules, those molecules need not be covalently bound, but it is possible for them to be covalently bound. When the two strands are covalently bound by means other than an uninterrupted strand of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the other strand, that binding structure is referred to as a "linker". The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs within the double strand is the number obtained by subtracting any overhangs present within the double strand from the number of nucleotides in the shortest strand of the dsRNA. In addition to the double-stranded structure, RNAi may include one or more nucleotide overhangs.

[0026] In other embodiments, the sense and antisense strands that hybridize to form the double-stranded region may be part of a single RNA molecule, i.e., the sense and antisense strands are part of a self-complementary region of a single RNA molecule. In such cases, the single RNA molecule includes a double-stranded region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is ligated to the 5' end of the antisense strand by an adjacent sequence of unpaired nucleotides to form the loop region. The loop region is typically long enough so that the RNA molecule itself can refold such that the antisense strand can base pair with the sense strand to form a duplex or stem region. The loop region can include from about 3 to about 25, from about 5 to about 15, or from about 8 to about 12 unpaired nucleotides. As described herein, such RNA molecules having at least partially self-complementary regions are referred to as "short hairpin RNAs" (shRNAs). In some embodiments, the loop region can include at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In other embodiments, the loop region can include 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer unpaired nucleotides. In certain embodiments, the RANi constructs disclosed herein include shRNAs. The length of a single at least partially self-complementary RNA molecule can be from about 35 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 85 nucleotides, or from about 50 to about 60 nucleotides, and can include a double-stranded region and a loop region having the lengths recited herein, respectively.

[0027] In some embodiments, the RNAi constructs disclosed herein include a sense strand and an antisense strand, and the antisense strand includes a region having a sequence that is substantially or completely complementary to the SCAP messenger RNA (mRNA) sequence. As used herein, the "SCAP mRNA sequence" refers to any messenger RNA sequence, including splice variants, that encodes a SCAP protein, where the SCAP protein includes variants or isoforms of the SCAP protein from any species (e.g., mouse, rat, non-human primate, human). SCAP is also known in the art as the SREBF chaperone.

[0028] In addition, the SCAP mRNA sequence includes a transcript sequence expressed as its complementary DNA (cDNA) sequence. The cDNA sequence refers to the sequence of an mRNA transcript represented as DNA bases (e.g., guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil, and cytosine). Thus, the antisense strand of the RNAi constructs disclosed herein may include a region having a sequence that is substantially or completely complementary to the target SCAP mRNA sequence or SCAP cDNA sequence. The SCAP mRNA or cDNA sequence may include, but is not limited to, any SCAP mRNA or cDNA sequence, such as those that may be derived from NCBI reference sequences NM_001320044.2 or NM_012235.4.

[0029] The region of the antisense strand can be substantially or completely complementary to at least 15 consecutive nucleotides of the SCAP mRNA sequence. In some embodiments, the target region of the SCAP mRNA sequence that the antisense strand containing the complementary region encompasses can be about 15 to about 30 consecutive nucleotides, about 16 to about 28 consecutive nucleotides, about 18 to about 26 consecutive nucleotides, about 17 to about 24 consecutive nucleotides, about 19 to about 25 consecutive nucleotides, about 19 to about 23 (e.g., 19, 20, 21, 22, or 23) consecutive nucleotides, or about 19 to about 21 consecutive nucleotides. In certain embodiments, the region of the antisense strand containing a sequence substantially or completely complementary to the SCAP mRNA sequence can, in some embodiments, contain at least 19 consecutive nucleotides from the antisense sequences listed in Table 1. In some embodiments, the sense sequence and / or the antisense sequence contain at least 15 consecutive nucleotides (e.g., at least 16, 17, or 18 consecutive nucleotides) having 1, 2, or 3 or fewer nucleotide mismatches from the sequences listed in Table 1.

[0030] The sense strand of an RNAi construct typically contains a sequence that is sufficiently complementary to the sequence of the antisense strand such that the two strands hybridize under physiological conditions to form a double-stranded region. The "double-stranded region" refers to a region within two complementary or substantially complementary polynucleotides that base pair with each other either by Watson-Crick base pairing or other hydrogen-bonding interactions to create a duplex between the two polynucleotides. The double-stranded region of an RNAi construct should be of a sufficient length, for example, such that binding of a Dicer enzyme and / or RISC complex (described below) enables the RNAi construct to enter the RNA interference pathway. For example, in some embodiments, the double-stranded region is about 15 to about 30 base pairs in length. Other lengths of the double-stranded region within this range, 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 are also suitable. In one embodiment, the double-stranded region is about 17 to about 24 base pairs in length. In another embodiment, the double-stranded region is about 19 to about 21 base pairs in length. For example, the double-stranded region may be about 19 base pairs in length.

[0031] In some embodiments, the RNAi constructs disclosed herein contain a double-stranded region of about 17 to about 24 nucleotides that interacts with a target RNA sequence, e.g., a SCAP target mRNA sequence, to direct cleavage of the target RNA. Without being bound by theory, long double-stranded RNA introduced into cells can be degraded to siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The ribonuclease III-like enzyme, Dicer, processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic 2-base 3’ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex to allow the complementary antisense strand to mediate target recognition (Nykanen, et al., (2001) Cell 107:309). As soon as it binds to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0032] For embodiments where the sense and antisense strands are two separate molecules (e.g., siRNA RNAi constructs), the sense and antisense strands need not be the same length as the length of the double-stranded region. For example, one or both strands may be longer than the double-stranded region and may have one or more unpaired nucleotides or mismatches adjacent to the double-stranded region. Thus, in some embodiments, the RNAi construct includes at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to unpaired nucleotides at the end of a strand or nucleotides that extend beyond the double-stranded region. 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 the 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 includes 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, the nucleotide overhang includes 1 to 4 nucleotides. In a certain embodiment, the nucleotide overhang includes 2 nucleotides. The nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some embodiments, the overhang includes a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide may include ribonucleotides or modified nucleotides, such as 2'-modified nucleotides. In other embodiments, the overhang includes a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide.

[0033] Nucleotide overhangs may be present at the 5' or 3' end of one or both strands. For example, in one embodiment, the RNAi construct includes nucleotide overhangs at the 5' and 3' ends of the antisense strand. In another embodiment, the RNAi construct includes nucleotide overhangs at the 5' and 3' ends of the sense strand. In some embodiments, the RNAi construct includes nucleotide overhangs at the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNAi construct includes nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.

[0034] The RNAi construct may include a single nucleotide overhang at one end of the double-stranded RNA molecule and blunt ends at the other end. A "blunt end" means that the sense and antisense strands are completely base-paired at the end of the molecule and there are no unpaired nucleotides extending beyond the double-stranded region. In some embodiments, the RNAi construct includes 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, the RNAi construct includes 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, the RNAi construct includes 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 double-stranded region is the same length as the sense and antisense strands (i.e., the molecule is double-stranded over its entire length).

[0035] The sense strand and the antisense strand can each independently be of any suitable length, for example, 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 are of the same length such that, to have an RNAi construct with a two-nucleotide overhang, they form a double-stranded region that is shorter than these strands. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each 21 nucleotides in length, (ii) a double-stranded region 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 double-stranded region 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 double-stranded region such that there are no nucleotide overhangs at either end of the double-stranded molecule over their entire length. In such an embodiment, the RNAi construct has blunt ends and comprises (i) a sense strand and an antisense strand each 21 nucleotides in length, and (ii) a double-stranded region 21 base pairs in length. In another embodiment, the RNAi construct has blunt ends and comprises (i) a sense strand and an antisense strand each 23 nucleotides in length, and (ii) a double-stranded region 23 base pairs in length.

[0036] In other embodiments, the sense or antisense strand is longer than the other strand such that the RNAi construct includes at least one nucleotide overhang, and the two strands form a double-stranded region having a length equal to the length of the shorter strand. For example, in one embodiment, the RNAi construct includes (i) a sense strand that is 19 nucleotides in length, (ii) an antisense strand that is 21 nucleotides in length, (iii) a double-stranded region that is 19 base pairs in length, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct includes (i) a sense strand that is 21 nucleotides in length, (ii) an antisense strand that is 23 nucleotides in length, (iii) a double-stranded region that is 21 base pairs in length, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand.

[0037] The antisense strand of the RNAi construct disclosed herein can include the sequence of any one of the antisense sequences listed in Table 1 or the sequence of nucleotides 1-18, 2-18, 1-19, 2-19, 1-21, or 2-21 of any of these antisense sequences. Each of the antisense sequences listed in Table 1 includes a sequence of 16-19 consecutive nucleotides complementary to the SCAP mRNA sequence in addition to the 2-nucleotide overhang sequence. Thus, in some embodiments, the antisense strand includes the sequence of nucleotides 1-18, 2-18, 1-19, 2-19, 1-21, or 2-21 of any one of SEQ ID NOs: 148-294 or SEQ ID NOs: 442-588. Similarly, the sense strand of the RNAi construct disclosed herein can include the sequence of any one of the sense sequences listed in Table 1 or the sequence of nucleotides 1-18, 2-18, 1-19, 2-19, 1-21, or 2-21 of any of these sense sequences. Thus, in some embodiments, the sense strand includes the sequence of nucleotides 1-18, 2-18, 1-19, 2-19, 1-21, or 2-21 of any one of SEQ ID NOs: 1-147 or SEQ ID NOs: 295-441.

[0038] Modified nucleotide The RNAi constructs disclosed herein, such as those listed in Table 1, 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 sensitivity of the molecule 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.

[0039] In certain embodiments, the modified nucleotide has a modification of the ribose sugar. These sugar modifications can include modifications at the 2' and / or 5' positions of the pentose ring and bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring with a substituent other than H or OH at the 2' position. Such 2' modifications include 2'-O-alkyl (e.g., O-C1-C10 or O-C1-C10 substituted alkyl), 2'-O-allyl (O-CH 2 CH=CH 2 ), 2'-C-allyl, 2'-fluoro, 2'-O-methyl (OCH 3 ), 2'-O-methoxyethyl (O-(CH 2 )) 2 OCH 3 ), 2'-OCF 3 , 2'-O(CH 2 )) 2 SCH 3, 2'-O-aminoalkyl, 2'-amino (e.g., NH 2 ), 2'-O-ethylamine, and 2'-azide, but are not limited thereto. 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.

[0040] "Bicyclic sugar modification" refers to a modification of the pentose ring that forms a second ring by linking two atoms of the ring by a bridge to produce a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification includes a bridge between the 4'-carbon and the 2'-carbon of the pentose ring. Nucleotides containing a sugar moiety having a bicyclic sugar modification are referred to herein as "bicyclic nucleic acids", "bridged nucleic acids", or "BNA". "Locked nucleic acid" (LNA) is a 2',4'-bicyclic nucleic acid (2',4'-BNA) in which the ribose ring is locked by a methylene bridge connecting the 2'-oxygen and the 4'-carbon. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4'-CH 2 -O-2') bicyclic nucleic acid (BNA); β-D-methyleneoxy (4'-CH 2 -O-2') BNA (LNA); ethyleneoxy (4'-(CH 2 )2-O-2') BNA; aminooxy (4'-CH 2 -O-N(R)-2') BNA; oxyamino (4'-CH 2 -N(R)-O-2') BNA; methyl (methyleneoxy) (4'-CH(CH 3 )-O-2') BNA (also referred to as constrained ethyl or cEt); methylene-thio (4'-CH 2 -S-2') BNA; methylene-amino (4'-CH 2 -N(R)-2') BNA; methyl carbocyclic (4'-CH 2 -CH(CH3)-2') BNA; propylene carbocyclic (4'-(CH 2 )3-2') BNA; and methoxy (ethyleneoxy) (4'-CH(CH 2Examples include, but are not limited to, OMe)-O-2’)BNA (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the RNAi constructs disclosed herein are described, for example, in U.S. Patent No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, 19:937-954 (2012).

[0041] 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 (BNA), 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 particular embodiment, the RNAi construct comprises one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof.

[0042] Both the sense and antisense strands of the RNAi construct can include one or more modified nucleotides. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In certain embodiments, all nucleotides within the sense strand are modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In other embodiments, all nucleotides of the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides can be 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof.

[0043] In certain embodiments, the modified nucleotides incorporated into one or both strands of the RNAi construct of the invention have a modification of the nucleobase (also referred to herein as "base"). A "modified nucleobase" or "modified base" refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases may be synthetic or naturally occurring modifications, and include 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-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine, but are not limited thereto.

[0044] In some embodiments, the modified base is a universal base. A "universal base" refers to a base analog that forms base pairs indiscriminately with all of the natural bases in RNA and DNA without altering the double helix structure of the resulting double-stranded region. Universal bases are known to those of skill in the art and include 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, but are not limited thereto.

[0045] Other suitable modified bases that can be incorporated into the RNAi constructs 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. Chem., Vol. 76:7295-7300, 2011, both of which are incorporated herein by reference in their entirety. Those skilled in the art are well aware that guanine, cytosine, adenine, thymine, and uracil can be replaced with other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of the polynucleotide containing nucleotides having such substituted nucleobases.

[0046] In some embodiments, the sense and antisense strands of the RNAi construct can include one or more abasic nucleotides. An “abasic nucleotide” or “abasic nucleoside” is a nucleotide or nucleoside that lacks a nucleobase at the 1’ position of the ribose sugar. In certain embodiments, the abasic nucleotide is incorporated at the end of the sense and / or antisense strand of the RNAi construct. In one embodiment, the sense strand includes an abasic nucleotide as the terminal nucleotide at its 3’ end, its 5’ end, or both its 3’ and 5’ ends. In another embodiment, the antisense strand includes an abasic nucleotide as the terminal nucleotide at its 3’ end, its 5’ end, or both its 3’ and 5’ ends. In such embodiments where the abasic nucleotide is the terminal nucleotide, the nucleotide can be an inverted nucleotide, i.e., it can be linked to an adjacent nucleotide via a 3’-3’ nucleotide linkage (when on the 3’ end of the strand) or a 5’-5’ nucleotide linkage (when on the 5’ end of the strand), rather than the natural 3’-5’ nucleotide linkage. Also, the abasic nucleotide can include a sugar modification, such as any of the sugar modifications described above. In certain embodiments, the abasic nucleotide includes a 2’-modification such as a 2’-fluoro modification, a 2’-O-methyl modification, or a 2’-H (deoxy) modification. In one embodiment, the abasic nucleotide includes a 2’-O-methyl modification. In another embodiment, the abasic nucleotide includes a 2’-H modification (i.e., a deoxy abasic nucleotide).

[0047] In some embodiments, in the sense strand and / or the antisense strand, all pyrimidine nucleotides preceding an adenosine nucleotide are modified nucleotides. For example, if 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 within the sense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides), and the 5' nucleotide of all sequences 5'-CA-3' or 5'-UA-3' present within the antisense strand is a modified nucleotide (e.g., 2'-O-methyl modified nucleotide). In other embodiments, all nucleotides within the double-stranded region are modified nucleotides. In such embodiments, the modified nucleotides are preferably 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or combinations thereof.

[0048] In embodiments where the RNAi construct comprises nucleotide overhangs, the nucleotides within the overhangs can be ribonucleotides, deoxyribonucleotides, or modified nucleotides. In one embodiment, the nucleotides within the overhangs are deoxyribonucleotides, for example, deoxythymidine. In another embodiment, the nucleotides within the overhangs are modified nucleotides. For example, in some embodiments, the nucleotides within the overhangs are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, or combinations thereof.

[0049] The RNAi constructs of the present disclosure may also include one or more modified nucleotide linkages. As used herein, the term "modified nucleotide linkage" refers to a linkage between nucleotides other than the natural 3'-5' phosphodiester linkage. In some embodiments, the modified nucleotide linkage is a phosphotriester, aminoalkyl phosphotriester, alkyl phosphonate (e.g., methyl phosphonate, 3'-alkylene phosphonate), phosphinate, phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkyl phosphoramidate), phosphorothioate (P=S), chiral phosphorothioate, phosphorodithioate, thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and boranophosphate, etc., phosphorus-containing nucleotide linkages. In one embodiment, the modified nucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, the modified nucleotide linkage is a phosphorus-free nucleotide linkage and thus may be referred to as a modified nucleoside linkage. Such phosphorus-free linkages include morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane linkages (-O-Si(H) 2 -O-); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (-CH 2 -N(CH 3 )-O-CH 2 -) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and N, O, S, and CH 2Other things in which the constituent parts are mixed can be mentioned, but are not limited to these. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) such as those described in U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262 for creating peptide nucleic acids or PNAs. Other suitable modified inter-nucleotide and internucleoside linkages that can be used in the disclosed RNAi constructs are described in U.S. Patent Nos. 6,693,187 and 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, supra.

[0050] In certain embodiments, the RNAi construct comprises one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages can 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 can 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, 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, 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, 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 (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 3′ end of the antisense strand) at the 3′ end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3′ end and 5′ end of the antisense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3′ end and 5′ end 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., at both the 5' and 3' ends of the antisense strand, including phosphorothioate internucleotide linkages at the first and second internucleotide linkages, and at both the 5' and 3' ends of the sense strand, including phosphorothioate internucleotide linkages at the first and second internucleotide linkages). In any of the embodiments where one or both strands contain one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages within the strand can be natural 3'-5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage of the sense and antisense strands is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0051] In embodiments where the RNAi construct comprises nucleotide overhangs, two or more of the unpaired nucleotides within the overhang can be linked by phosphorothioate internucleotide linkages. In certain embodiments, all of the unpaired nucleotides in the nucleotide overhang at the 3' end of the antisense strand and / or the sense strand are linked by phosphorothioate internucleotide linkages. In other embodiments, all of the unpaired nucleotides in the nucleotide overhang at the 5' end of the antisense strand and / or the sense strand are linked by phosphorothioate internucleotide linkages. In yet other embodiments, all of the unpaired nucleotides within any nucleotide overhang are linked by phosphorothioate internucleotide linkages.

[0052] In some embodiments, the 5' end of the sense strand, the antisense strand, or both the antisense and sense strands of the disclosed RNAi construct comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to terminal phosphate groups including unmodified phosphate (-O-P=O)(OH)OH) as well as modified phosphates. Modified phosphates include phosphates in which one or more of the O and OH groups are substituted with H, O, S, N(R) or alkyl, where R is H, an amino protecting group or an unsubstituted or substituted alkyl. Exemplary phosphate moieties include 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or non-methylated); 5'-adenosine cap or any other modified or unmodified nucleotide cap structure; 5'-monothiophosphate (phosphorothioate); 5'-monodithiophosphate (phosphorodithioate); 5'-α-thiotriphosphate; 5'-γ-thiotriphosphate; 5'-phosphoramidate; 5'-vinyl phosphate; 5'-alkyl phosphonate (where "alkyl" can be methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonate (where "alkyl ether" can be methoxymethyl, ethoxymethyl, etc.), but are not limited thereto.

[0053] The modified nucleotides that can be incorporated into the RNAi constructs disclosed herein can have two or more chemical modifications as described herein. For example, the modified nucleotide can have a modification of the ribose sugar as well as a modification of the nucleobase. By way of example, the modified nucleotide may include a 2'-sugar modification (e.g., 2'-fluoro or 2'-O-methyl), and may include a modified base (e.g., 5-methylcytosine or pseudouridine). In other embodiments, the modified nucleotide may include a sugar modification combined with a modification to the 5'-phosphate, such that when the modified nucleotide is incorporated into a polynucleotide, it will result in modified internucleotide or inter-nucleoside linkages. For example, in some embodiments, the modified nucleotide can 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 the RNAi construct include a combination of 2'-modified nucleotides or BNA and phosphorothioate nucleotide linkages. In certain embodiments, both the sense and antisense strands of the RNAi construct include a combination of 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, and phosphorothioate nucleotide linkages. Exemplary RNAi constructs containing modified nucleotides and internucleotide linkages are shown in Tables 1 and 2.

[0054] Function of the RNAi construct The RNAi constructs disclosed herein desirably reduce or inhibit the expression of SCAP in cells, particularly liver cells. Thus, in one embodiment, the present disclosure provides a method of reducing SCAP expression in a cell by contacting the cell with any of the RNAi constructs described herein. The cell may be in vitro or in vivo. SCAP expression can be evaluated by measuring the amount or level of SCAP mRNA, SCAP protein, or another biomarker associated with SCAP expression. Reduction of SCAP expression in a cell or animal treated with an RNAi construct disclosed herein can be determined by comparison to SCAP expression in a cell or animal not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, reduction of SCAP expression is evaluated by: (a) measuring the amount or level of SCAP mRNA in liver cells treated with an RNAi construct disclosed herein; (b) measuring the amount or level of SCAP mRNA in liver cells treated with a control RNAi construct (e.g., an RNAi construct directed to an RNA molecule not expressed in liver cells, or an RNAi construct having a nonsense or scrambled sequence), or no construct; and (c) comparing the measured SCAP mRNA level from the cells treated in (a) to the measured SCAP mRNA level from the control cells in (b). The SCAP mRNA levels in the treated and control cells may be normalized for RNA level with respect to a control gene (e.g., 18S ribosomal RNA) prior to comparison. SCAP mRNA levels can be measured by various methods including Northern blot analysis, nuclease protection assay, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, and quantitative PCR.

[0055] In other embodiments, reduction of SCAP expression is evaluated by: (a) measuring the amount or level of SCAP protein in liver cells treated with an RNAi construct disclosed herein; (b) measuring the amount or level of SCAP protein in liver cells treated with a control RNAi construct (e.g., an RNAi construct directed to an RNA molecule not expressed in liver cells, or an RNAi construct having a nonsense or scrambled sequence), or without a construct; and (c) comparing the measured SCAP protein level from the cells treated in (a) to the measured SCAP protein level from the control cells in (b). SCAP protein levels can be measured using any suitable method known to those of skill in the art, including but not limited to Western blot, immunoassay (e.g., ELISA), and flow cytometry. The effectiveness of the RNAi constructs disclosed herein can be evaluated using any suitable method for measuring SCAP mRNA or protein.

[0056] In some embodiments, the method for evaluating SCAP expression level is performed in vitro in cells that naturally express SCAP (e.g., liver cells) or cells engineered to express SCAP. In certain embodiments, the method is performed in vitro in liver cells. Suitable liver cells include, but are not limited to, primary hepatocytes (e.g., human, non-human primate, or rodent hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the liver cells are Hep3B cells. In another embodiment, the liver cells are HepG2 cells.

[0057] In other embodiments, the method for assessing SCAP expression levels is performed in vivo. For example, an RNAi construct and any control RNAi construct can be administered to an animal (e.g., a rodent or non-human primate), and the mRNA or protein levels of SCAP can be evaluated in liver tissue collected from the treated animal. Alternatively, or in addition, biomarkers or functional phenotypes associated with SCAP expression can be evaluated in the treated animal.

[0058] In certain embodiments, the expression of SCAP is reduced by at least 40%, at least 45%, or at least 50% in liver cells by the RNAi constructs disclosed herein. In some embodiments, the expression of SCAP is reduced by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% in liver cells by the RNAi constructs disclosed herein. In other embodiments, the expression of SCAP is reduced by about 90% or more, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more in liver cells by the RNAi constructs disclosed herein. The percent reduction in the expression of SCAP can be measured by any of the methods described herein or other methods known in the art. For example, in certain embodiments, the RNAi construct inhibits at least 40% of SCAP expression at 5 nM in Hep3B cells (containing wild-type SCAP) in vitro. In related embodiments, the RNAi construct inhibits at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of SCAP expression at 5 nM in Hep3B cells in vitro. In other embodiments, the RNAi construct inhibits at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of SCAP expression at 5 nM in Hep3B cells in vitro. In certain embodiments, the RNAi constructs disclosed herein inhibit at least 40% of SCAP expression at 5 nM in HepG2 cells in vitro. In related embodiments, the RNAi constructs disclosed herein inhibit at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of SCAP expression at 5 nM in HepG2 cells in vitro.In other embodiments, the RNAi construct inhibits at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of SCAP expression at 5 nM in HepG2 cells in vitro. In certain embodiments, the RNAi construct inhibits at least 40% of SCAP expression at 5 nM in CHO transfected cells expressing human SCAP in vitro. In related embodiments, the RNAi construct inhibits at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of SCAP expression at 5 nM in CHO transfected cells expressing human SCAP in vitro. In other embodiments, the RNAi construct inhibits at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of SCAP expression at 5 nM in CHO transfected cells expressing human SCAP in vitro. Reduction of SCAP can be measured using a variety of techniques including, for example, RNA FISH or droplet digital PCR (see, e.g., Kamitaki et al., Digital PCR. Methods in Molecular Biology, 1768:401-422 (2018). doi:10.1007 / 978-1-4939-7778-9_23).

[0059] In some embodiments, IC 50 values are calculated to evaluate the potency of the RNAi constructs disclosed herein against inhibition of SCAP expression in liver cells. The "IC 50 value" is the dose / concentration required to achieve 50% inhibition of a biological or biochemical function. In other embodiments, the potency of the RNAi construct can be evaluated by calculating the "AC 50 value", which is the dose / concentration required to achieve 50% activation of a biological or biochemical function. The IC 50 value or AC 50Values can be determined by constructing a dose - response curve in any assay and testing the effects of various concentrations of a substance or antagonist on expression levels or functional activity. By determining the concentration required to inhibit half of the maximal biological response or the original expression level, the IC 50 value of a given antagonist or substance can be calculated. Thus, in any assay such as an immunoassay, an RNA FISH assay, or a droplet digital PCR assay, by determining the concentration of an RNAi construct required to inhibit half of the original expression level of SCAP in liver cells (e.g., the expression level of SCAP in control liver cells), the IC 50 value for any RNAi construct can be calculated. Similarly, by determining the concentration required to activate half of the maximal biological response or the original expression level, the AC 50 value of a given substance can be calculated. The RNAi constructs disclosed herein can inhibit SCAP expression in liver cells (e.g., Hep3B cells) with an IC 50 of less than about 20 nM (e.g., less than about 15 nM, 10 nM, 5 nM, or 1 nM). For example, the disclosed RNAi constructs can inhibit SCAP expression in liver cells with an IC 50 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 liver cells (e.g., Hep3B cells) with an IC 50 of about 1 nM to about 10 nM (e.g., about 5 nM). The RNAi constructs disclosed herein have an IC 50can inhibit SCAP expression in liver cells (e.g., HepG2 cells). For example, the RNAi construct has an IC 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 50 can inhibit SCAP expression in liver cells. In certain embodiments, the RNAi construct has an IC of about 1 nM to about 10 nM (e.g., about 5 nM) 50 inhibits SCAP expression in liver cells (e.g., HepG2 cells). The RNAi constructs disclosed herein have an IC of less than about 20 nM 50 can inhibit SCAP expression in cells expressing human SCAP (e.g., cells transfected with CHO). For example, the RNAi construct has an IC 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 50 inhibits SCAP expression in SCAP-expressing cells. In certain embodiments, the RNAi construct has an IC of about 1 nM to about 10 nM (e.g., about 5 nM) 50 inhibits SCAP expression in SCAP-expressing cells.

[0060] The RNAi constructs disclosed herein can be readily prepared using techniques known in the art such as conventional nucleic acid solid-phase synthesis. The polynucleotides of the RNAi construct can be constructed using suitable nucleic acid synthesizers with standard nucleotides 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).

[0061] Oligonucleotides can be synthesized via phosphoramidite chemistry using a 2'-silyl protecting group together with an 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 a large, medium, or small scale using any automated or manual synthesizer. Also, the synthesis can be carried out in multiple well plates, columns, or glass slides.

[0062] 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 an inorganic counterion, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with an organic counterion, such as tetraalkylammonium fluoride. Crown ether catalysts can be utilized in combination with inorganic fluoride in the deprotection reaction. Exemplary fluoride ion sources include, but are not limited to, tetrabutylammonium fluoride or amino hydrofluoride (e.g., by combining triethylamine and aqueous HF in an aprotic solvent such as dimethylformamide).

[0063] The stability of the triesters to fluoride can be modified by the choice of protecting groups used on the phosphite triesters and phosphotriesters. Methyl protection of the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve the process yield.

[0064] Since ribonucleosides have a reactive 2'-hydroxyl substituent, it may be desirable to protect the reactive 2'-position in RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, such as one that is stable to acid treatment. Silyl protecting groups meet this condition and can be readily removed in the final fluoride deprotection step, thereby minimizing RNA degradation.

[0065] In a standard phosphoramidite coupling reaction, a tetrazole catalyst can be used. Exemplary catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0066] Additional methods for synthesizing the RNAi constructs described herein will be apparent to those of skill in the art. Additionally, the various synthetic steps may be carried out in alternative orders or sequences to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on bases) and protecting group manipulations (protection and deprotection) useful in the synthesis of the RNAi constructs described herein are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), as well as their later editions. 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).

[0067] The RNAi constructs disclosed herein may include a ligand. As used herein, "ligand" refers to any compound or molecule that can interact directly or indirectly with another compound or molecule. The interaction between another compound or molecule and the ligand may elicit a biological response (e.g., initiate a signal transduction cascade, induce receptor-mediated endocytosis), or it may be just a physical association. The ligand can modify one or more properties of the double-stranded RNA molecule to which it binds, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, intracellular uptake, charge, and / or clearance properties of the RNA molecule.

[0068] The ligand may include serum proteins (e.g., human serum albumin, low density lipoprotein, globulin), cholesterol moieties, vitamins (e.g., biotin, vitamin E, vitamin B12), folic acid moieties, steroids, bile acids (e.g., cholic acid), fatty acids (e.g., palmitic acid, myristic acid), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), glycosides, phospholipids or antibodies or their binding fragments (e.g., whole antibodies or binding fragments targeting RNAi constructs for specific cell types such as liver cells). Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptides (e.g., antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers (e.g., polyethylene glycol (PEG), e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0069] In certain embodiments, the ligand has endosome destabilizing properties. An endosome destabilizing ligand promotes the lysis of endosomes and / or the transport of an RNAi construct or a component thereof from the endosome to the cytoplasm of the cell. The endosome destabilizing ligand can be a polycationic peptide or peptidomimetic that exhibits pH-dependent membrane activity and membrane fusogenicity. In one embodiment, the endosome destabilizing ligand adopts its active conformation at the pH of the endosome. The "active" conformation is the conformation of the endosome destabilizing ligand that promotes the lysis of endosomes and / or the transport of an RNAi construct or a component thereof from the endosome to the cytoplasm of the cell. Exemplary endosome destabilizing ligands include the GALA peptide (Subbarao et al., Biochemistry, Vol. 26:2964-2972, 1987), the EALA peptide (Vogel et al., J. Am. Chem. Soc., Vol. 118:1581-1586, 1996) and derivatives thereof (Turk et al., Biochem. Biophys. Acta, Vol. 1559:56-68, 2002). In one embodiment, the endosome destabilizing component can contain a chemical group (e.g., an amino acid) that will undergo a change in charge or protonation in response to a change in pH. The endosome destabilizing component can be linear or branched.

[0070] 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 acids are also described in U.S. Patent Nos. 7,851,615; 7,745,608; and 7,833,992. In another embodiment, the ligand may comprise a folic acid moiety. Polynucleotides conjugated to folic acid moieties can be taken up by cells via receptor-dependent endocytosis pathways. Such folic acid-polynucleotide conjugates are described, for example, in U.S. Patent No. 8,188,247.

[0071] Given that SCAP is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it may be desirable to specifically deliver an RNAi construct to liver cells. In some embodiments, the RNAi construct can be specifically targeted to the liver by employing a ligand that binds or interacts with a protein expressed on the surface of liver cells. For example, in certain embodiments, the ligand may comprise one or more antigen-binding proteins (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically bind to a receptor expressed on hepatocytes.

[0072] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound composed of one or more monosaccharide units (which can be linear, branched, or cyclic) having at least six carbon atoms to which an oxygen, nitrogen, or sulfur atom is bonded to each carbon atom. 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), as well as 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.

[0073] In some embodiments, the ligand comprises a hexose or a hexosamine. The hexose can be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine can 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 a particular embodiment, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose and N-acetyl-galactosamine (GalNAc) are particularly effective at targeting compounds to liver cells (see, e.g., D’Souza and Devarajan, J. Control Release, Vol. 203:126-139, 2015). Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs disclosed herein are described in U.S. Patent Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Application Publication No. 2003 / 0130186; and International Publication No. 2013 / 166155 pamphlet.

[0074] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, "multivalent carbohydrate moiety" refers to a moiety that includes two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety can include two or more binding domains composed of carbohydrates that can bind to two or more different molecules, or two or more different sites on the same molecule. The valency of the carbohydrate moiety indicates 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 1, 2, 3, and 4 binding domains, respectively. The multivalent carbohydrate moiety can include a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety is divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be bifurcated or trifurcated. In one particular embodiment, the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary GalNAc-containing ligands for incorporation into the RNAi constructs disclosed herein include trifurcated GalNAc-containing ligands (also referred to as "GalNAc3").

[0075] The ligand can be directly or indirectly bound or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is covalently directly bound to the sense strand or the antisense strand of the RNAi construct. In other embodiments, the ligand is covalently bound to the sense strand or the antisense strand of the RNAi construct via a linker. The ligand can be bound to the nucleobase, sugar moiety or internucleotide linkage of the polynucleotide (e.g., sense strand or antisense strand) of the RNAi construct disclosed herein. Conjugation or binding to a purine nucleobase or its derivative can occur at any position including intra-ring atoms and exo-ring 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 its derivative can also occur at any position. In some embodiments, the 2-, 5- and 6-positions of the pyrimidine nucleobase can be bound to the ligand. Conjugation or binding to the sugar moiety of the nucleotide can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety to which the ligand can be bound include the 2’, 3’ and 5’ carbon atoms. For example, in a depurinated nucleotide, the 1’ position can also be bound to the ligand. The internucleotide linkage can also support the binding of the ligand. In the case of a phosphorus-containing linkage (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand can be directly bound to the phosphorus atom or can be bound to an O, N or S atom bound to the phosphorus atom. In the case of an amine- or amide-containing internucleoside linkage (e.g., PNA), the ligand can be bound to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0076] In certain embodiments, the ligand may be attached to either 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. For example, in some embodiments, the ligand is attached to the 5’ terminal nucleotide of the sense strand. In such certain embodiments, the ligand is attached at the 5’ position of the 5’ terminal nucleotide of the sense strand. In other embodiments, the ligand is covalently attached to the 3’ end of the sense strand. For example, in some embodiments, the ligand is attached to the 3’ terminal nucleotide of the sense strand. In such certain embodiments, the ligand is attached at the 3’ position of the 3’ terminal nucleotide of the sense strand. In alternative embodiments, the ligand is near the 3’ end of the sense strand but is attached prior to (i.e., before) one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2’ position of the sugar of the 3’ terminal nucleotide of the sense strand.

[0077] In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. A "linker" is an atom or group of atoms that covalently attaches the ligand to the polynucleotide component of the RNAi construct. The linker may 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 include a bifunctional linking moiety that generally includes an alkyl moiety having two functional groups. One of the functional groups is selected to bind to the compound of interest (e.g., the sense or antisense strand of the RNAi construct), and the other is selected to bind substantially to any selected group, such as a ligand as described herein. In certain embodiments, the linker includes a chain structure or oligomer consisting of repeating units such as ethylene glycol units or amino acid units. Examples of functional groups typically used 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, examples of the bifunctional linking moiety include amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double bond or triple bond), and the like.

[0078] Linkers that can be used to attach a ligand to the sense or antisense strand of an RNAi construct include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C 1 ~C 10 alkyl, substituted or unsubstituted C 2 ~C 10 alkenyl or substituted or unsubstituted C 2 ~C 10Examples of the alkynyl include, but are not limited to, these. 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.

[0079] In certain embodiments, the linker is cleavable. A cleavable linker is sufficiently stable outside the cell but is cleaved upon entry into the target cell, releasing 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, 90-fold, or at least 100-fold faster in the target cell or under a first reference condition (e.g., selected to mimic or correspond to intracellular conditions) than in the subject's blood or under a second reference condition (e.g., selected to mimic or correspond to conditions found in blood or serum).

[0080] A cleavable linker is sensitive to a cleavage agent, such as the presence of pH, redox potential, or a degradable molecule. Generally, the cleavage agent is predominant or found at high levels or activities inside the cell rather than in serum or blood. Examples of such degradable agents include redox agents, such as oxidoreductases (selected for a particular substrate or substrate-nonspecific), e.g., oxidases or reductases, or reducing agents, such as mercaptans, that are present intracellularly and can decompose a redox-cleavable linker by reduction; esterases; agents that can generate an endosome or an acidic environment, e.g., those that bring about a pH of 5 or less; enzymes, peptidases (which may be substrate-specific), and phosphatases that can hydrolyze or decompose an acid-cleavable linker by acting as a general acid.

[0081] Cleavable linkers can contain a pH-sensitive moiety. The pH of human serum is 7.4, while the average intracellular pH is slightly lower than that, in the range of about 7.1 - 7.3. Endosomes have a more acidic pH in the range of 5.5 - 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will release the RNA molecule from the ligand into the interior of the cell or into a desired compartment of the cell by having a cleavable group that is cleaved at a preferred pH.

[0082] Linkers can contain a cleavable group that is cleavable by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the cell being targeted. For example, a liver-targeting ligand can be attached to an RNA molecule via a linker containing an ester group. Liver cells are rich in esterases, and thus the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis. When targeting cells rich in peptidases, such as liver cells and synoviocytes, a linker containing a peptide bond can be used.

[0083] Generally, the suitability of a cleavable linker candidate can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the linker candidate. It is also desirable to test a cleavable linker candidate for its ability to resist cleavage when in contact with blood or other non-target tissues. Thus, the relative sensitivity to cleavage can be determined between a first condition selected to show cleavage within the target cell and a second condition selected to show cleavage within other tissues or body fluids, such as blood or serum. The evaluation can be performed in a cell-free system, in cells, cell cultures, organ or tissue cultures or in whole animals. It can be useful to perform an initial evaluation under cell-free or culture conditions and confirm by further evaluation in whole animals. In some embodiments, a useful linker candidate is 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) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0084] 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 group (-S-S-). One or more of the methods described herein can be used to determine whether a cleavable linker candidate is a suitable "reductively cleavable linker" or is suitable for use, for example, with a particular RNAi construct and a particular ligand. For example, a linker candidate can be evaluated by incubating it with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate that would be observed in cells such as target cells. The linker candidate can also be evaluated under conditions selected to mimic blood conditions or serum conditions. In certain embodiments, the linker candidate is cleaved by no more than 10% in blood. In other embodiments, a useful linker candidate is degraded at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in cells (or in vitro conditions selected to mimic intracellular conditions) compared to blood (or in vitro conditions selected to mimic extracellular conditions).

[0085] In yet other embodiments, a phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that hydrolyze phosphate groups intracellularly include enzymes such as intracellular phosphatases. Examples of phosphate-based cleavable groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S-, where Rk is hydrogen or C 1 ~C 10It can be alkyl. Specific embodiments include -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -SP(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. Another specific embodiment is -O-P(O)(OH)-O-. These linker candidates can be evaluated using methods similar to those described above.

[0086] In other embodiments, the linker can 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 at a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent such as an enzyme that can act as a general acid. Inside cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid-cleavable group can have the general formula -C=NN-, C(O)O, or -OC(O). A specific embodiment is when 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.

[0087] In other embodiments, the linker may include an ester-based cleavable group that is cleaved by enzymes such as esterases and amidases in the cell. 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.

[0088] In further embodiments, the linker may include a peptide-based cleavable group that is cleaved by enzymes such as peptidases and proteases in the cell. Peptide-based cleavable groups are peptide bonds formed between amino acids that result in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids that results in peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that result in peptides and proteins and do not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula -NHCHR A C(O)NHCHR B C(O)-, wherein R A and R B are the side chains of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0089] Other types of linkers suitable for attaching ligands to the sense or antisense strand in the RNAi constructs described herein are known in the art and can include, for example, the linkers described in U.S. Patent Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551.

[0090] In certain embodiments, the ligand covalently attached to the sense or antisense strand of the RNAi construct 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 still 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.

[0091] In some embodiments, the RNAi constructs disclosed herein can be delivered to a target cell or tissue by administering a vector that encodes and controls the intracellular expression of the RNAi construct. A "vector" (also referred to herein as an "expression vector") is a composition of matter that can be used to deliver a nucleic acid of interest into the interior of a cell. A number of vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides complexed with 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, retroviral vectors, etc. The vector may be replicated in a living cell or may be synthetically produced.

[0092] Generally, a vector for expressing an RNAi construct will contain one or more promoters operably linked to a sequence encoding the RNAi construct. The phrases "operably linked," "operatively linked," or "under transcriptional control" may be used interchangeably herein to indicate that the promoter is in the correct position and orientation relative to the polynucleotide sequence to initiate transcription by RNA polymerase and control the expression of the polynucleotide sequence. A "promoter" refers to a sequence that is recognized by or introduced into the cellular synthetic apparatus and is required to initiate transcription of a particular gene sequence. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI, or U6 RNA pol III, and viral promoters (e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the long terminal repeat of Rous sarcoma virus). In some embodiments, the HI or U6 RNA pol III promoter is utilized. The promoter can be a tissue-specific promoter or an inducible promoter. Of particular note are liver-specific promoters such as promoter sequences derived from the human α1-antitrypsin gene, albumin gene, hemopexin gene, and hepatic lipase gene. Inducible promoters include, for example, promoters regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-β-D-1-thiogalactopyranoside (IPTG).

[0093] When the RNAi construct contains siRNA, two separate strands (sense strand and antisense strand) can be expressed from a single vector or two separate vectors. For example, in some embodiments, the sequence encoding the sense strand is operably linked to a promoter of a first vector, and the sequence encoding the antisense strand is operably linked to a promoter of a second vector. In such embodiments, the first and second vectors are introduced into the target cells simultaneously, for example, by infection or transfection, so that when the sense strand and antisense strand are transcribed, they hybridize intracellularly to form siRNA molecules. In another embodiment, the sense strand and antisense strand are transcribed from two separate promoters located within a single vector. In such embodiments, the sequence encoding the sense strand may be operably linked to a first promoter, and the sequence encoding the antisense strand may be operably linked to a second promoter, and the first and second promoters are arranged within a single vector. In one embodiment, the vector includes a first promoter operably linked to a sequence encoding an siRNA molecule and a second promoter operably linked in the reverse direction to the same sequence, such that transcription of the sequence from the first promoter results in the synthesis of the sense strand of the siRNA molecule and transcription of the sequence from the second promoter results in the synthesis of the antisense strand of the siRNA molecule.

[0094] When the RNAi construct contains shRNA, 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 shRNA includes inverted repeats joined by a linker polynucleotide sequence, which generates the stem and loop structure of shRNA after transcription.

[0095] In some embodiments, the vector encoding the RNAi construct is a viral vector. Suitable viral vector systems for expressing the RNAi constructs described herein include, but are not limited to, adenoviral vectors, retroviral vectors (e.g., lentiviral vectors, Moloney murine leukemia virus), adeno-associated viral vectors; herpes simplex virus vectors; SV40 vectors; polyomavirus vectors; papillomavirus vectors; picornavirus vectors; and poxvirus vectors (e.g., vaccinia virus). In certain embodiments, the viral vector is a retroviral vector (e.g., a lentiviral vector).

[0096] Methods for inserting nucleic acid sequences encoding various vectors, siRNA or shRNA molecules suitable for use in the present disclosure into vectors, and methods for delivering the vectors to target cells are known in the art (see, for example, Dornburg, Gene Therap., Vol. 2: 301-310, 1995; Eglitis, Biotechniques, Vol. 6: 608-614, 1988; Miller, Hum Gene Therap., Vol. 1: 5-14, 1990; Anderson, Nature, Vol. 392: 25-30, 1998; Rubinson D A et al., Nat. Genet., Vol. 33: 401-406, 2003; Brummelkamp et al., Science, Vol. 296: 550-553, 2002; Brummelkamp et al., 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., Genes Dev, 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).

[0097] Composition The present disclosure also provides 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 the expression of SCAP in a subject in need of reducing the expression of SCAP. When considering clinical use, the pharmaceutical compositions and formulations will be prepared in a form appropriate for the intended use. Generally, this will involve the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0098] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier, excipient, or diluent" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are acceptable for use in the formulation of pharmaceuticals such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. All conventional media or agents are intended to be used in the therapeutic compositions, except in cases where they are incompatible with the RNAi constructs of the present disclosure. Auxiliary active ingredients can also be incorporated into the compositions, provided they do not inactivate the vector or RNAi construct of the composition.

[0099] Compositions and methods for formulating pharmaceutical compositions depend on several criteria including, but not limited to, the route of administration, the type and extent of the disease or disorder to be treated, and the dosage to be administered. In some embodiments, the pharmaceutical composition is formulated based on the intended delivery route. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, and intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such embodiments, the pharmaceutical composition may comprise a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such embodiments, the pharmaceutical composition may comprise a targeting ligand (e.g., a GalNAc-containing ligand described herein).

[0100] In some embodiments, the pharmaceutical composition comprises an effective amount of an RNAi construct described herein. An "effective amount" is an amount sufficient to produce an advantageous or desired clinical result. In some embodiments, the effective amount is an amount sufficient to reduce SCAP expression in the hepatocytes of a subject. In some embodiments, the effective amount may be an amount sufficient to only partially reduce SCAP expression to a level comparable to the expression of the wild-type SCAP allele in a human heterozygote, for example.

[0101] The effective amount of the RNAi construct disclosed herein can be from about 0.01 mg / kg body weight to about 100 mg / kg body weight, from about 0.05 mg / kg body weight to about 75 mg / kg body weight, from about 0.1 mg / kg body weight to about 50 mg / kg body weight, from about 1 mg / kg to about 30 mg / kg body weight, from about 2.5 mg / kg body weight to about 20 mg / kg body weight, or from about 5 mg / kg body weight to about 15 mg / kg body weight. The pharmaceutical composition containing the effective amount of the RNAi construct can be administered weekly, bi-weekly, monthly, quarterly, or semi-annually. Accurately determining the dosage and frequency of administration that are considered effective can be based on several factors including the size, age, sex of the patient, the type of disorder to be treated (e.g., obesity, NAFLD, NASH, or cirrhosis), the specific RNAi construct used, and the route of administration. The estimated effective dosage and in vivo half-life of any specific RNAi construct disclosed herein can be confirmed using conventional methods and / or testing in appropriate animal models.

[0102] Colloidal dispersion systems such as polymer composites, nanocapsules, microspheres, beads, and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the RNAi constructs disclosed herein or vectors encoding such constructs. Commercially available fat emulsions suitable for nucleic acid delivery include INTRALIPID®, LIPOSYN®, LIPOSYN® II, LIPOSYN® III, NUTRILIPID, and other similar fat emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is liposomes (i.e., artificial membrane vesicles). The RNAi construct can be encapsulated within liposomes such as cationic liposomes. Alternatively, the RNAi constructs disclosed herein can be complexed with lipids, such as cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems are well known in the art. Exemplary formulations are also disclosed, for example, in U.S. Patent Nos. 5,783,565; 5,837,533; 5,981,505; 6,127,170; 6,217,900; 6,379,965; 6,383,512; 6,747,014; 7,202,227; and International Publication No. 03 / 093449 pamphlet.

[0103] In some embodiments, the RNAi constructs disclosed herein are fully encapsulated in a lipid formulation to form, for example, SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles including SPLP. As used herein, the term "SPLP" refers to nucleic acid-lipid particles containing plasmid DNA encapsulated in lipid vesicles. SNALP and SPLP typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particles (e.g., a PEG-lipid conjugate). SNALP and SPLP exhibit a long circulation lifetime after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site), and thus are very useful for systemic administration. SPLP includes "pSPLP" that includes an encapsulated condensing agent-nucleic acid complex as described in WO 00 / 03683 pamphlet. The 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. In addition, the nucleic acid present in the nucleic acid-lipid particles desirably is resistant to nuclease degradation in an aqueous solution. The nucleic acid-lipid particles and methods for preparing the same are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432, and WO 96 / 40964 pamphlet.

[0104] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In general, these preparations are sterile and are fluid to the extent that they are readily injectable. The preparations must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media can contain, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by using coatings (such as lecithin), by maintaining the required particle size (in the case of dispersions), and / or by using surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents (such as sugars or sodium chloride) can be included in the composition. Sustained absorption of the injectable composition can be brought about by including absorption delaying agents, such as aluminum monostearate and gelatin.

[0105] Sterile injectable solutions can be prepared by incorporating a suitable amount of the RNAi construct (alone or complexed with a ligand) into a solvent, optionally together with any other ingredients as described above, followed by filtration sterilization. In general, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the desired other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying techniques and freeze-drying techniques, from which powders of the active ingredient plus any additional desired ingredients are obtained from its pre-sterilized filtered solution.

[0106] The compositions provided herein can be formulated in the neutral form or in the form of a salt. Pharmaceutically acceptable salts include, for example, 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, mandelic acid, etc.). Also, salts formed with free carboxyl groups can be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0107] For parenteral administration in aqueous solution, for example, the solution is generally suitably buffered and the liquid diluent is first made isotonic, for example, with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. As is known to those skilled in the art, it is desirable to use a sterile aqueous medium. As an example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected into the indicated injection site (see, for example, “Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035 - 1038 and 1570 - 1580). For human administration, the preparation must meet the standards of sterility, pyrogenicity, general safety, and purity required by FDA standards. In certain embodiments of the present disclosure, the pharmaceutical composition comprises or consists of sterile saline and the RNAi construct described herein. In other embodiments, the pharmaceutical composition comprises or consists of the RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet other embodiments, the pharmaceutical composition comprises or consists of the RNAi construct described herein and phosphate buffered saline (PBS).

[0108] In some embodiments, the pharmaceutical composition is packaged in or stored within a dosing device. Examples of devices for injectable formulations include, but are not limited to, injection ports, prefilled syringes, automatic infusion devices, infusion pumps, wearable syringes, and injection pens. Examples of devices for aerosolized or powder formulations include, but are not limited to, inhalers, nebulizers, aspirators, and the like. Accordingly, the present disclosure includes a dosing device comprising a pharmaceutical composition for treating or preventing one or more of the disorders described herein.

[0109] Method for inhibiting SCAP expression The present disclosure also provides a method for inhibiting the expression of the SCAP gene in a cell. The method includes contacting the cell with an RNAi construct, such as a double-stranded RNAi construct, in an amount effective to inhibit the expression of SCAP in the cell. Contacting the cell with an RNAi construct, such as a double-stranded RNAi construct, can be performed in vitro or in vivo. Contacting the cell with an RNAi construct in vivo includes contacting a cell or cell population within a subject, such as a human subject, with the RNAi construct. Combinations of in vitro and in vivo methods of contacting the cell are also within the scope of the present disclosure.

[0110] The present disclosure provides a method for reducing or inhibiting the expression of SCAP in a subject in need thereof, and a method for treating or preventing a symptom, disease, or disorder associated with SCAP expression or activity. "Symptom, disease, or disorder associated with SCAP expression" refers to a symptom, disease, or disorder in which the level of SCAP expression changes or an increase in the level of SCAP expression is associated with an increased risk of onset of the symptom, disease, or disorder.

[0111] Contacting the cells may be direct or indirect as discussed above. Further, contacting the cells may be achieved via a targeting ligand that includes any ligand described herein or known in the art. In a preferred embodiment, the targeting ligand is a carbohydrate moiety such as a tri-branched GalNAc ligand (GalNAc3) or any other ligand that directs an RNAi construct to the site of interest.

[0112] In one embodiment, contacting the cells with the RNAi includes "introducing" or "delivering the RNAi to the cells" by promoting or causing uptake or absorption into the cells. Absorption or uptake of the RNAi can occur by passive diffusivity or active cellular processes without assistance, or by an assisting agent or device. For example, for in vivo introduction, the RNAi may be injected into the tissue site or systemically administered. In vitro introduction into cells can be achieved using methods known in the art such as electroporation and lipofection. Further methods are described herein below and / or are known in the art.

[0113] As used herein, the term "inhibit" is used interchangeably with "reduce", "silence", "down-regulate", "suppress", and other similar terms and includes any level of inhibition.

[0114] The phrase "inhibiting the expression of SCAP" is intended to refer to the inhibition of the expression of any SCAP gene (e.g., the mouse SCAP gene, the rat SCAP gene, the monkey SCAP gene, or the human SCAP gene) as well as variants or mutants of the SCAP gene. Thus, the SCAP gene can be a wild-type SCAP gene, a mutant SCAP gene, or a transgenic SCAP gene in the context of a genetically engineered cell, cell population, or organism.

[0115] "Inhibiting the expression of the SCAP gene" includes inhibiting the SCAP gene at any level, for example, suppressing at least a part of the expression of the SCAP gene. The expression of the SCAP gene can be evaluated based on the level of any variable associated with SCAP gene expression, such as the SCAP mRNA level or the SCAP protein level, or the change in the level. This level can be evaluated, for example, in individual cells or cell populations including samples derived from a subject.

[0116] Inhibition can be evaluated by a decrease in the absolute or relative level of one or more variables associated with SCAP expression as compared to a control level. The control level can be any type of control level utilized in the art, such as, for example, a baseline level before administration, or a level determined from a similar subject, cell, or sample treated with no treatment or a control (e.g., a buffer-only control or an inactive agent control). In some embodiments, the 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%.

[0117] Inhibition of SCAP gene expression can be demonstrated by a decrease in the amount of mRNA expressed by a first cell or cell population that is substantially identical to, but not similarly treated as, a second cell or cell population (control cells) and in which SCAP gene transcription is inhibited and the cells are treated (e.g., by contacting the cells with an RNAi construct disclosed herein or by administering an RNAi construct disclosed herein to the subject in which the cells are present or were present). Such cells can be present, for example, in a sample derived from a subject. The inhibition can be evaluated by expressing the mRNA level in the treated cells as a percentage of the mRNA level in the control cells using the following formula.

Number

[0118] In addition, inhibition of SCAP gene expression can be evaluated in terms of a reduction in parameters that are functionally related to SCAP gene expression, such as SCAP protein expression or SREBP protein family activity. SCAP gene silencing can be determined in any cell that endogenously or recombinantly expresses SCAP by any assay known in the art.

[0119] Inhibition of SCAP protein expression can be demonstrated by a reduction in the level of SCAP protein expressed by a cell or cell population (e.g., the level of protein expressed in a sample obtained from a subject). As described above, inhibition of protein expression levels in treated cells or cell populations can be similarly expressed as a percentage of the protein level in control cells or cell populations to evaluate mRNA suppression.

[0120] As a control cell or cell group that can be used to evaluate the inhibition of SCAP gene expression, cells or cell groups that have not yet come into contact with the RNAi construct disclosed herein can be mentioned. For example, the control cell or control cell group may be obtained from an individual subject (e.g., a human subject or an animal subject) prior to treatment of the subject with the RNAi construct.

[0121] The level of SCAP mRNA expressed by a cell or cell group, or the level of circulating SCAP mRNA, can be determined using any method known in the art for evaluating mRNA expression, such as the methods described above. In some embodiments, the level of SCAP expression in a sample is determined by detecting a transcriptional polynucleotide or a part thereof, such as the mRNA of the SCAP gene. In this regard, for example, 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 assay, RT-PCR, RNase protection assay (Melton et al., Nuc. Acids Res., 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating SCAP mRNA can be detected using the method described in WO 2012 / 177906 pamphlet.

[0122] 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 sequence. The probe can be synthesized by one skilled in the art or derived from a suitable biological preparation. The probe may be specially designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0123] Isolated mRNA can be used in hybridization or amplification assays including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining the mRNA level involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA of SCAP. In one embodiment, for example, the isolated mRNA is electrophoresed on an agarose gel and transferred from the gel to a membrane such as nitrocellulose to immobilize the mRNA on a solid surface and then contacted with the probe. In an alternative embodiment, the probe is immobilized on a solid surface, for example, in an Affymetrix gene chip array, and the mRNA is contacted with the probe. One skilled in the art can readily adapt known mRNA detection methods for use in determining the level of SCAP mRNA.

[0124] Alternative methods for determining the expression level of SCAP in a sample include, for example, RT-PCR (see, e.g., U.S. Patent 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 system (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., supra; and U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, including, for example, nucleic acid amplification of mRNA in a sample and / or the process of reverse transcriptase, followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when they are present in very small numbers. In some aspects of the present disclosure, the expression level of SCAP can be determined by quantitative fluorescence-generating RT-PCR (i.e., the TAQMAN (trademark) system). The expression level of SCAP mRNA can be monitored using membrane blot (such as those used in hybridization analysis such as Northern, Southern, dot, etc.) or using microwells, sample tubes, gels, beads or fibers (or any solid support containing bound nucleic acid) (see, e.g., U.S. Patent Nos. 5,445,934; 5,677,195; 5,770,722; 5,744,305; and 5,874,219). Determination of the SCAP expression level may include the use of nucleic acid probes in solution. In certain embodiments, the level of mRNA expression is evaluated using branched DNA (bDNA) assay or real-time PCR (qPCR).

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

[0126] In some embodiments, the effectiveness of the methods disclosed herein can be monitored by detecting or monitoring a reduction in symptoms of SCAP-related diseases such as gastrointestinal pain, dyspnea, hypertension, or swelling of the limbs, face, larynx, upper airway, abdomen, conductor, and genitalia. These symptoms can be evaluated in vitro or in vivo using any method known in the art.

[0127] In some aspects, an RNAi construct or a composition comprising 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 evaluated using measurement of the level or change in 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 some embodiments, the RNAi construct can be delivered to sites such as the liver, choroid plexus, retina, and pancreas. The site can be a small unit or subgroup of cells derived from any one of the aforementioned sites. Additionally, the site can include cells that express a specific type of receptor.

[0128] Method for treating or preventing SCAP-related diseases The present disclosure provides therapeutic and prophylactic methods comprising administering to a subject having or prone to developing a SCAP-related disease, disorder and / or condition, an RNAi construct, a composition comprising the RNAi construct (e.g., a pharmaceutical composition), or a vector comprising the RNAi construct described herein. Non-limiting examples of SCAP-related diseases include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, liver inflammation, hepatocyte necrosis, liver fibrosis, obesity, and non-alcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and hyperlipidemia. 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.

[0129] In certain embodiments, the present disclosure provides a method for reducing the expression of SCAP in a patient in need thereof, the method comprising administering to the patient any of the RNAi constructs described herein. As used herein, the term "patient" refers to a mammal including a human and can be used interchangeably with the term "subject". It is desirable that the expression level of SCAP in the hepatocytes of the patient be reduced after administration of the RNAi construct as compared to the SCAP expression level of a patient not receiving the RNAi construct.

[0130] The methods disclosed herein are useful for treating subjects suffering from a SCAP-related disease, e.g., subjects who would benefit from a reduction in SCAP gene expression and / or SCAP protein production. In one aspect, the present disclosure provides a method for reducing the level of SCAP gene expression in a subject suffering from non-alcoholic fatty liver disease (NAFLD). In another aspect, the present disclosure provides a method for reducing the level of SCAP protein in a subject having NAFLD.

[0131] The treatment methods (and uses) disclosed herein involve administering to a subject, such as a human, a therapeutically effective amount of an RNAi construct that targets the SCAP gene, a pharmaceutical composition comprising the RNAi construct, or a vector comprising the RNAi construct.

[0132] In one aspect, the present disclosure provides a method for preventing at least one symptom in a subject having NAFLD, such as the presence of an enhanced hedgehog signaling pathway, fatigue, debilitation, weight loss, anorexia, nausea, abdominal pain, spider veins, yellowing of the skin and eyes (jaundice), itching, fluid retention and leg swelling (edema), abdominal distension (ascites), and confusion. The method involves administering to the subject a prophylactically effective amount of an RNAi construct, such as siRNA, a pharmaceutical composition comprising the RNAi construct, or a vector encoding the RNAi construct, thereby preventing at least one symptom in a subject having a disorder that would benefit from reduced SCAP gene expression. "Prophylactically effective amount" refers to an amount effective at the required dosage and for the period of time necessary to achieve the desired prophylactic result, e.g., prevention of disease onset.

[0133] In another aspect, the present disclosure provides the use of a therapeutically effective amount of an RNAi construct disclosed herein for treating a subject, such as a subject that would benefit from reduced and / or inhibited SCAP gene expression. In a further aspect, the present disclosure provides the use of an RNAi construct that targets the SCAP gene, such as siRNA, or a pharmaceutical composition comprising an RNAi construct that targets the SCAP gene, in the manufacture of a medicament for treating a subject, such as a subject having a disorder, e.g., a subject having an SCAP-related disease, that would benefit from reduced and / or inhibited SCAP gene expression and / or SCAP protein production.

[0134] The present disclosure provides the use of RNAi constructs, such as the siRNAs disclosed herein, to prevent at least one symptom in a subject suffering from a disorder that would benefit from a reduction and / or inhibition of SCAP gene expression and / or SCAP protein production. For example, the present disclosure provides the use of the RNAi constructs described herein, compositions comprising the same, and vectors comprising the same, in the treatment of NAFLD.

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

[0136] In one embodiment, the RNAi construct targeting SCAP is such that when the RNAi construct is administered to a subject, the expression of the SCAP gene is reduced 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%, 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 in a subject having an SCAP-related disease, such as non-alcoholic fatty liver disease (NAFLD).

[0137] The methods and uses disclosed herein include administering a composition described herein such that expression of a target SCAP gene is reduced at any suitable time, 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 by the methods and uses disclosed herein can result in a reduction in the severity, signs, symptoms, and / or markers of such a disease or disorder in a patient having an SCAP-related disease, such as NAFLD. "Reduction" in this context means a statistically significant decrease at 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 treating or preventing a disease can be evaluated, for example, by measuring disease progression, disease remission, severity of the condition, reduction of pain, quality of life, the dosage of the pharmaceutical required to maintain the therapeutic effect, the level of a disease marker, or any other measurable parameter suitable for a given disease being treated or targeted for prevention. Observation of the effectiveness of treatment or prevention by measuring any one or any combination of such parameters is well within the ability of one of ordinary skill in the art. For example, the effectiveness of treating NAFLD can be evaluated, for example, by the periodic monitoring of NAFLD symptoms, liver fat levels, or the expression of downstream genes. Comparison of an initial reading with a later reading provides an indication to the physician as to whether the treatment is effective. In connection with the administration of an RNAi targeting SCAP or a pharmaceutical composition thereof, "effective against" an SCAP-related disease means that administration in a clinically appropriate manner results in beneficial effects such as improvement of the condition, cure, reduction of the disease, prolongation of life, improvement of the quality of life, or other effects generally recognized as favorable by a physician skilled in the treatment of NAFLD and / or SCAP-related diseases and related causes for at least a statistically significant proportion of patients.

[0139] A therapeutic or prophylactic effect is demonstrated when there is a statistically significant improvement in one or more parameters of the disease state, or when the symptoms that would otherwise be expected do not worsen or develop. By way of example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more of a measurable parameter of the disease may mean an effective treatment. The effectiveness of a given RNAi construct or a formulation of that construct can 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 the treatment is demonstrated when a statistically significant reduction in a marker or symptom is observed.

[0140] Any therapeutically effective amount of an RNAi construct can be administered to a subject. Exemplary ranges of a therapeutically effective amount of an RNAi construct include, but are not limited to, from about 0.01 mg / kg body weight to about 100 mg / kg body weight, from about 0.05 mg / kg body weight to about 75 mg / kg body weight, from about 0.1 mg / kg body weight to about 50 mg / kg body weight, from about 1 mg / kg to about 30 mg / kg body weight, or from about 2.5 mg / kg body weight to about 20 mg / kg body weight. Intermediate values and ranges within the recited ranges are also included in the present disclosure.

[0141] Administration of an RNAi construct, or a composition comprising the same, can reduce the presence of SCAP protein levels in, for example, a patient's cells, tissues, blood, urine, or other compartments 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%, or by at least about 99% or more.

[0142] Prior to administration of the total dose of RNAi, a smaller dose, such as 5% of the infusion solution, may be administered to the patient to monitor for adverse effects such as allergic reactions. In another example, the patient may be observed for unwanted immunostimulatory effects such as an increase in cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0143] Due to the inhibitory effect on SCAP expression, the composition according to the present disclosure, or a pharmaceutical composition prepared therefrom, can improve the quality of life of a patient suffering from an SCAP-related disease (e.g., NAFLD).

[0144] The RNAi disclosed herein may be administered in “naked” form, in which case the modified or unmodified RNAi construct is suspended directly as “free RNAi” in an aqueous or suitable buffered solvent. Free RNAi is administered in the absence of a pharmaceutical composition. Free RNAi may be in a suitable buffered solution. The buffered solution may contain acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and weight osmolarity of the buffered solution containing RNAi can be adjusted to be suitable for administration to a subject.

[0145] Alternatively, the RNAi constructs disclosed herein can be administered as a pharmaceutical composition, such as a liposomal formulation.

[0146] Subjects who would benefit from a reduction and / or inhibition of SCAP gene expression are subjects having non-alcoholic fatty liver disease (NAFLD) and / or another SCAP-related disease or disorder as described herein or otherwise known in the art.

[0147] The present disclosure further provides a method for treating a subject who would benefit from a reduction and / or inhibition of SCAP gene expression, e.g., a subject having an SCAP-related disease, by combining an RNAi construct or a pharmaceutical composition thereof with other pharmaceuticals and / or other therapies, such as those currently used for treating these disorders, e.g., known pharmaceuticals and / or known therapies, and also provides the use of the RNAi construct or its pharmaceutical composition.

[0148] For example, in certain embodiments, an RNAi construct that targets the SCAP gene is administered in combination with, for example, an agent useful for treating an SCAP-related disease. For example, additional therapeutic agents and therapies suitable for treating a subject who would benefit from reduced SCAP expression, e.g., a subject suffering from an SCAP-related disease, include RNAi constructs that target different portions of the SCAP gene, therapeutic agents and / or procedures for treating an SCAP-related disease, or any combination of the foregoing.

[0149] In certain embodiments, a first RNAi construct that targets a portion of the SCAP gene is administered in combination with a second RNAi construct that targets a different portion of the SCAP gene. For example, the first RNAi construct may include a first sense strand and a first antisense strand that form a duplex region, substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand being modified nucleotides, the first sense strand being conjugated to a ligand attached to the 3' end, the ligand being one or more GalNAc derivatives attached via a divalent or trivalent branched linker; and the second RNAi construct can include a second sense strand and a second antisense strand that form a duplex region, substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand being modified nucleotides, the second sense strand being conjugated to a ligand attached to the 3' end, the ligand being one or more GalNAc derivatives attached via a divalent or trivalent branched linker. 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 include modifications. The modified nucleotides can be any one or combination of the modified nucleotides described herein.

[0150] In other embodiments, a first RNAi construct targeting the SCAP gene is administered in combination with a second RNAi construct targeting a gene different from the SCAP gene. For example, an RNAi construct targeting the SCAP gene may be administered in combination with an RNAi construct targeting the patatin-like phospholipase domain containing the 3 (PNPLA3) gene. The I148M mutant PNPLA3 protein is a therapeutic target with strong human gene validation for the treatment of non-alcoholic steatohepatitis (NASH). Expression of PNPLA3 I148M results in the accumulation of excessive liver fat and causes non-alcoholic fatty liver disease (NAFLD)-related phenotypes with increased incidence, progression, and severity. The first RNAi construct targeting the SCAP gene and a second RNAi construct targeting a different gene, e.g., the PNPLA3 gene, may be administered as part of the same pharmaceutical composition. Alternatively, the first RNAi construct targeting the SCAP gene and a second RNAi construct targeting a different gene, e.g., the PNPLA3 gene, may be administered as part of different pharmaceutical compositions.

[0151] The RNAi construct and additional therapeutic agent(s) 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 another method known in the art or described herein.

[0152] The present disclosure also provides methods of using the RNAi constructs and / or compositions containing the RNAi constructs disclosed herein to reduce and / or inhibit SCAP expression (gene or protein expression) in cells. In yet other aspects, there is provided the use of the RNAi and / or compositions containing RNAi disclosed herein for the manufacture of a medicament for reducing and / or inhibiting SCAP gene expression in cells. In still other aspects, the present disclosure provides compositions containing the RNAi and / or RNAi constructs described herein for use in reducing and / or inhibiting SCAP protein production in cells. In yet other aspects, there is provided the use of the RNAi constructs and / or compositions containing the RNAi constructs described herein for the manufacture of a medicament for reducing and / or inhibiting SCAP protein production in cells. The methods and uses involve contacting the cells with an RNAi construct disclosed herein, such as siRNA, and maintaining the cells for a time sufficient to obtain degradation of the mRNA transcript of the SCAP gene, thereby inhibiting the expression of the SCAP gene in the cells or inhibiting SCAP protein production. Reduction of gene expression can be evaluated by any method known in the art or described herein for determining mRNA or protein levels.

[0153] In the methods and uses disclosed herein, the cells may be contacted in vitro or in vivo, i.e., the cells may be outside the subject (e.g., during cell culture) or within the subject. Suitable cells for treatment using the methods disclosed herein 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 comprising the SCAP gene or a portion of the SCAP gene. Suitable cells for use in the methods of the present disclosure include, for example, mammalian cells, such as primate cells (human cells or non-human primate cells, such as monkey cells or chimpanzee cells, etc.), non-primate cells (bovine cells, porcine cells, camel cells, llama cells, equine cells, goat cells, rabbit cells, sheep cells, hamster, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells or buffalo cells, etc.), avian cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cells are human cells.

[0154] SCAP gene expression can be inhibited 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%, or about 100% in the cell.

[0155] SCAP protein production can be inhibited 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%, or about 100% in cells.

[0156] The in vivo methods and uses disclosed herein may include administering a composition containing an RNAi construct, which contains a nucleotide sequence complementary to at least a portion of the RNA transcript of the SCAP gene of the subject. When the organism to be treated is a human, the composition may be administered by any means known in the art, such as parenteral routes including subcutaneous, intravenous, oral, intraperitoneal or intracranial (e.g., intraventricular, intrenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, as well as local (including buccal and sublingual) administration, but not limited thereto. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection. In one embodiment, the composition is administered by subcutaneous injection.

[0157] In some embodiments, administration is by depot injection. Depot injections can release the RNAi construct consistently over a long period of time. Thus, depot injections can reduce the frequency of administration required to obtain a desired effect, such as inhibition of a desired SCAP, or a therapeutic or prophylactic effect. Also, depot injections can achieve a more consistent serum concentration. Examples of depot injections can include subcutaneous injection or intramuscular injection. In some embodiments, the depot injection is a subcutaneous injection.

[0158] In some embodiments, administration is via a pump. The pump can 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. Infusion pumps can 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 the subject.

[0159] The mode of administration may be selected based on whether a local or systemic treatment is desired and also based on the area to be treated. The route of administration and the site of administration can be selected to enhance targeting.

[0160] The methods and uses include administering to a mammal, such as a human, a composition comprising an RNAi construct, such as an siRNA that targets the SCAP gene in mammalian cells, and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the SCAP gene, thereby inhibiting the expression of the SCAP gene in the mammal. Reduction of gene expression and / or protein expression can be evaluated in a sample obtained from a subject administered the RNAi construct by any method known in the art or described herein. In one embodiment, the tissue sample functions as tissue material for monitoring reduction of SCAP gene and / or protein expression. In another embodiment, the blood sample functions as tissue material for monitoring reduction of SCAP gene and / or protein expression.

[0161] In some embodiments, verification of RISC-mediated cleavage of target mRNA (e.g., SCAP mRNA) in vivo after administration of an RNAi construct can be evaluated by performing 5'-RACE or a modification of a protocol known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38(3) p-e19; and Zimmermann et al. (2006) Nature 441:111-4).

[0162] It is understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by substituting uracil bases in the sequence with thymine bases. Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by substituting thymine bases in the sequence with uracil bases. All deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing mixtures of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein are encompassed by this disclosure.

[0163] In addition, any nucleic acid sequence disclosed herein can be modified by any combination of chemical modifications. Those skilled in the art will readily understand that in certain cases, such designations as "RNA" or "DNA" for describing modified polynucleotides are arbitrary. For example, a polynucleotide containing nucleotides having a 2'-OH substituent on the ribose sugar and thymine bases can be described as a DNA molecule having a modified sugar (2'-OH relative to the natural 2'-H of DNA) or an RNA molecule having a modified base (thymine (methylated uracil) relative to the natural uracil of RNA).

[0164] Accordingly, the nucleic acid sequences provided herein, including but not limited to those set forth in the Sequence Listing, are intended to encompass nucleic acids containing such nucleic acids with modified nucleobases, including but not limited to any combination of natural or modified RNA and / or DNA containing such nucleic acids. As a further example, without limitation, a polynucleotide having the sequence "ATCGATCG" includes those having RNA bases, whether modified or unmodified, such as the sequence "AUCGAUCG", as well as those having some DNA bases and RNA bases such as "AUCGATCG", and polynucleotides having other modified bases such as "ATmeCGAUCG", and includes such compounds including any polynucleotide having such a sequence, where meC in the sequence represents a cytosine base containing a methyl group at the 5-position.

[0165] The following examples, including the experiments performed and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the appended claims.

Examples

[0166] All animal experiments described in this specification 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, 8th Edition (National Research Council (U.S.)). They were managed in accordance with the committee for the update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (U.S.) and National Academies Press (U.S.) (2011) Guide for the care and use of laboratory animals. 8th Ed., National Academies Press, Washington, D.C. Mice were housed individually in a temperature-controlled space at 22 ± 2 °C on a 12-hour light; 12-hour dark cycle (0600 - 1800 hours). Animals were provided with regular solid feed diet (Envigo, 2920X, or otherwise specified diet) and water (reverse osmosis purified) by an automatic watering system ad libitum, unless otherwise instructed. At the end, blood was collected by cardiac puncture under deep anesthesia, and then euthanized by secondary physical methods in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.

[0167] Example 1: Selection, Design, and Synthesis of Modified SCAP siRNA Molecules The identification and selection of optimal sequences for therapeutic siRNA molecules targeting the sterol regulatory element-binding protein cleavage-activating protein (SCAP) gene were confirmed using bioinformatics analysis of the human SCAP transcript. The siRNA trigger was prepared as a chemically modified siRNA duplex consisting of sense (passenger) and antisense (guide) strands. Both strands were 18 - 23 nucleotides in length with a 2-base pair 3'-overhang. The natural 2'-OH within the ribose of each nucleotide was replaced with either a 2'-OMe or 2'-F group, and the two phosphodiester nucleotide internucleotide linkages at each end of both strands were replaced with phosphorothioates to modify the siRNA to reduce degradation by endonucleases. The resulting unmodified and modified antisense as well as sense siRNA sequences are shown in Table 1.

[0168] The nucleotide sequences in Table 1 and other parts of this application are listed according to the following notations. A, U, G, and C = corresponding ribonucleotides; dT = deoxythymidine; dA = deoxyadenosine; dC = deoxycytidine; dG = deoxyguanosine; invAb = inverted abasic; invDT = inverted deoxythymidine; invDA = inverted deoxyadenosine; invDC = inverted deoxycytidine; invDG = inverted deoxyguanosine; a, u, g, and c = corresponding 2'-O-methyl ribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; Ab = abasic; MeO-I = 2'-methoxyinosine; GNA = glycol nucleic acid; sGNA = glycol nucleic acid with 3'-phosphorothioate; LNA = locked nucleic acid. The insertion of "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothioester group (e.g., phosphorothioate internucleotide linkage). Unless otherwise indicated, all other nucleotides are linked by 3'-5' phosphodiester groups.

[0169]

Table 1-1

[0170]

Table 1-2

[0171]

Table 1-3

[0172]

Table 1-4

[0173]

Table 1-5

[0174]

Table 1-6

[0175]

Table 1-7

[0176]

Table 1-8

[0177]

Table 1-9

[0178]

Table 1-10

[0179]

Table 1-11

[0180]

Table 1-12

[0181] Example 2: Efficacy of Selected SCAP siRNA Molecules in RNA FISH Assay The siRNA molecules synthesized in Example 1 were screened in a fluorescence in situ hybridization assay targeting ribonucleic acid molecules (RNA FISH), and IC 50 and the maximum activity value were determined.

[0182] An RNA FISH assay was performed to measure the knockdown of SCAP mRNA by the test siRNA. Hep3B (purchased from ATCC) was cultured in minimum essential medium (MEM, Corning) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (P-S, Corning). siRNA transfection was performed as follows: 1 μL of the test siRNA and 4 μL of plain MEM were added by a BioMek FX (Beckman Coulter) to a CellCarrier-384 Ultra assay plate (PerkinElmer) coated with PDL. Next, 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) (0.035 μL of RNAiMAX in 5 μL MEM) pre-diluted in plain MEM was dispensed into the assay plate by 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 (2000 cells per well) in MEM supplemented with 10% FBS and 1% P-S were added to the transfection complex using a Multidrop Combi Reagent Dispenser, and the assay plate was held at room temperature for 20 minutes and then moved to the incubator. The cells were incubated at 37 °C and 5% CO 2 for 72 hours.

[0183] The RNA FISH assay was performed according to the manufacturer's protocol using the Affymetrix QuantiGene® View RNA HC Screening Assay Kit (QVP0011), the Affymetrix View HC Signal Amplification Kit 3-plex (QVP0213), and Affymetrix gene-specific probes (SCAP Human 0.33mL View RNA Type 6 (650 labels) VA6-20279-01 and PPIB Human 0.44mL View RNA Type 1 (488 labels) VA1-10148-01).

[0184] First, the plates were rehydrated by sequential 100%, 70%, and 50% ethanol washes. Next, the cells were washed with PBS and then permeabilized and protease digested according to the kit instructions. The Working Probe Set of interest was prepared according to the manufacturer's protocol, added to the wells, and incubated at 40 °C for 3 hours. For sequential hybridization with the Working Probe Set, Working PreAmp, Working Amp, and Working LP, the manufacturer's protocol was followed. Finally, nuclear counterstaining was applied (Hoechst 33342 and Cell Mask Blue; Molecular Probes). The plates were incubated at room temperature for 30 minutes, washed with PBS, overlaid with 80 μl of PBS, and subsequently sealed for imaging.

[0185] All plates were imaged on an Opera Phenix high-content screening system (PerkinElmer) using the UV channel for Hoechst 33342 and Cell Mask Blue, the 488 channel for the Type1 probe, and the 647 channel for the Type6 probe.

[0186] RNA FISH data was analyzed using Columbus software, and images were generated using Genedata Screener. The images were analyzed to obtain the average number of spots per cell. The number of spots was normalized using high (containing phosphate-buffered saline, Corning) and low (without the target probe pair) control wells. Values normalized against the total siRNA concentration were plotted, and the data was fitted to a four-parameter sigmoid model in Genedata Screener (Genedata) to obtain the IC 50 and maximum activity.

[0187] The results of the assay are shown in Table 2. SCAP activity is expressed as the percentage of knockdown compared to the control. Negative values indicate a decrease in SCAP level. Additional tests were performed on several siRNA triggers, and their results are shown in the last column of Table 2.

[0188]

Table 2-1

[0189]

Table 2-2

[0190]

Table 2-3

[0191]

Table 2-4

[0192]

Table 2-5

[0193]

Table 2-6

[0194]

Table 2-7

[0195] Example 3: Screening for the efficacy of selected SCAP siRNA molecules in a mouse model Male Balbc mice 7-8 weeks old and weighing more than 20 grams were obtained from Charles River Laboratories (Charles River Laboratories, Inc, MA). The mice were administered an AAV8 viral vector for robust liver expression. Each animal was administered 1×10 12 viral particles reconstituted in 200 μl of cell culture grade PBS. The AAV vector encoded a reporter construct based on the luciferase gene. Luciferase activity was analyzed by measuring the light signal generated from the luciferin / luciferase reaction after a 10-minute intraperitoneal administration of the Xenolight Rediject D-luciferin substrate (Perkim Elmer). These light signals were shown as flux. Bioluminescence imaging (BLI) was performed using an IVIS Lumina S5 Spectrum preclinical in vivo imaging device from Perkin Elmer. BLI image analysis was performed by placing small identical regions of interest (ROIs) on the liver and analyzed using Living Image software 4.7.2.

[0196] To monitor SCAP siRNA activity, the 3’UTR region of the luciferase reporter gene contained a specific SCAP mRNA sequence to which the siRNA could bind and initiate degradation of the luciferase reporter gene. Two weeks after AAV administration, mice were imaged for baseline luciferase activity. The animals were then randomized according to baseline image intensity and grouped into five animals per siRNA. The next day, mice were administered SCAP siRNA conjugated to trisialylated GalNAc (GalNAc3) at the 5’ end of the sense strand subcutaneously at 3 mg per kg body weight (mpk). Four weeks after siRNA administration, BLI imaging was performed to monitor luciferase activity. The siRNA efficacy at week 4 was calculated as the ratio of the week 4 (Wk4) flux:baseline flux for each siRNA group and presented in Table 3 as the silencing % data normalized to the PBS group. A total of 158 triggers were tested, some of which contained different chemical modifications.

[0197]

Table 3-1

[0198]

Table 3-2

[0199]

Table 3-3

[0200]

Table 3-4

[0201]

Table 3-5

[0202]

Table 3-6

[0203]

Table 3-7

[0204] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference 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 references in this specification should not be construed as an admission that such references are prior art to the present invention. To the extent that any definition or term provided in a reference incorporated by reference is inconsistent with the terms and considerations provided herein, the terms and definitions provided herein shall control.

[0205] The foregoing specification is considered to be sufficient to enable those skilled in the art to practice the invention. The foregoing description and examples have set forth in detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. However, it will be understood that, however detailed the foregoing may be, the invention may be practiced in many ways and that the invention is to be construed in accordance with the appended claims and their equivalents.

Claims

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand includes a region having at least 15 consecutive nucleotides that differ from the antisense sequences listed in Table 1 by 3 nucleotides or less, and the RNAi construct inhibits the expression of SREBP cleavage-activating protein (SCAP) mRNA.

2. The RNAi construct according to claim 1, wherein the sense strand includes a sequence sufficiently complementary to the sequence of the antisense strand in order to form a double-stranded region of about 15 to about 30 base pairs in length.

3. The RNAi construct according to claim 1, wherein the double-stranded region is approximately 17 to approximately 24 base pairs long.

4. The RNAi construct according to claim 3, wherein the double-stranded region is approximately 19 to approximately 21 base pairs long.

5. The RNAi construct according to claim 4, wherein the double-stranded region is 19 base pairs long.

6. The RNAi construct according to claim 1, wherein the sense strand and the antisense strand are each about 15 to about 30 nucleotides in length.

7. The RNAi construct according to claim 6, wherein the sense strand and the antisense strand are each about 19 to about 27 nucleotides in length.

8. The RNAi construct according to claim 7, wherein the sense strand and the antisense strand are each about 21 to about 25 nucleotides in length.

9. The RNAi construct according to claim 8, wherein the sense strand and the antisense strand are each about 21 to about 23 nucleotides long.

10. The RNAi construct according to claim 1, comprising at least one blunt end.

11. The RNAi construct according to claim 1, comprising at least one nucleotide overhang of 1 to 4 unpaired nucleotides.

12. The RNAi construct according to claim 11, wherein the nucleotide overhang has two unpaired nucleotides.

13. The RNAi construct according to claim 12, wherein the RNAi construct includes a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or at the 3' ends of both the sense strand and the antisense strand.

14. The RNAi construct according to claim 13, wherein the nucleotide overhang comprises a 5'-UU-3' dinucleotide or a 5'-dTdT-3' dinucleotide.

15. The RNAi construct according to claim 1, wherein the RNAi construct comprises at least one modified nucleotide.

16. The RNAi construct according to claim 15, wherein the modified nucleotide is a 2'-modified nucleotide.

17. The RNAi construct according to claim 15, wherein the modified nucleotide is a 2'-fluoromodified 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 nucleotide, or a combination thereof.

18. The RNAi construct according to claim 17, wherein the modified nucleotide is a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-fluoro modified nucleotide, or a combination thereof.

19. The RNAi construct according to claim 15, wherein all of the nucleotides in the sense strand and the antisense strand are modified nucleotides.

20. The RNAi construct according to claim 19, wherein the modified nucleotide is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or a combination thereof.

21. The RNAi construct according to claim 1, comprising at least one phosphorothioate nucleotide interbonding.

22. The RNAi construct according to claim 21, wherein the RNAi construct includes two consecutive phosphorothioate nucleotide bonds at the 3' end of the antisense strand.

23. The RNAi construct according to claim 21, wherein the RNAi construct includes two consecutive phosphorothioate nucleotide bonds at both the 3' and 5' ends of the antisense strand, and includes two consecutive phosphorothioate nucleotide bonds at the 5' end of the sense strand.

24. The RNAi construct according to claim 1, wherein the antisense strand includes a sequence selected from the antisense sequences listed in Table 1.

25. The RNAi construct according to claim 1, wherein the sense strand includes a sequence selected from the sense sequences listed in Table 1.

26. The RNAi construct according to claim 1, wherein the RNAi construct is one of the double-stranded compounds listed in Table 1.

27. The RNAi construct according to claim 1, wherein the RNAi construct is a small interfering RNA (siRNA).

28. The RNAi construct according to claim 1, wherein the RNAi construct reduces the expression level of SCAP in liver cells after incubation with the RNAi construct compared to the expression level of SCAP in liver cells incubated with a control RNAi construct.

29. The RNAi construct according to claim 28, wherein the liver cells are Hep3B cells or HepG2 cells.

30. The RNAi construct according to claim 29, wherein the RNAi construct inhibits at least 40% of SCAP expression at 5 nM in in vitro Hep3B cells.

31. The RNAi construct according to claim 29, wherein the RNAi construct inhibits at least 40% of SCAP expression at 5 nM in in vitro HepG2 cells.

32. The RNAi construct according to claim 29, wherein the RNAi construct inhibits SCAP expression in Hep3B cells with an IC50 of less than approximately 5 nM.

33. The RNAi construct according to claim 29, wherein the RNAi construct inhibits SCAP expression in HepG2 cells with an IC50 of less than approximately 5 nM.

34. The RNAi construct according to claim 1, further comprising ligands that bind to one or more proteins expressed on the surface of liver cells.

35. A composition comprising the RNAi construct described in claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

36. A composition for reducing SCAP expression in patients requiring reduction of SCAP expression, comprising an RNAi construct according to any one of claims 1 to 34.

37. The composition according to claim 35 for reducing SCAP expression in patients who require a reduction in SCAP expression.

38. The composition according to claim 36, wherein the expression level of SCAP in hepatocytes is reduced in a patient after administration of the RNAi construct, compared to the SCAP expression level in a patient who has not received the RNAi construct.

39. The composition according to claim 36, wherein the patient suffers from non-alcoholic fatty liver disease (NAFLD).

40. The composition according to claim 39, wherein the patient suffers from non-alcoholic steatohepatitis (NASH).

41. A composition comprising the RNAi construct according to any one of claims 1 to 34 or the composition according to claim 35 for use in the treatment of NAFLD.

42. Use of an RNAi construct according to any one of claims 1 to 34 or a composition according to claim 35 for the preparation of a pharmaceutical product for treating NAFLD.