Rnai constructs for inhibiting SCAP expression and methods of using same

Abstract

The present disclosure relates to RNAi constructs, such as siRNAs, for reducing the expression of the SCAP gene. Also described are methods of treating or preventing liver diseases, such as non-alcoholic fatty liver disease (NAFLD), using such RNAi constructs.

Description

Technical Field

[0001] The present disclosure relates to compositions and methods for regulating the hepatic expression of SREBP cleavage activation protein (SCAP). In particular, the present disclosure relates to nucleic acid-based therapeutic agents that reduce SCAP expression via RNA interference, and methods of using such nucleic acid-based therapeutic agents to treat or prevent liver diseases such as non-alcoholic fatty liver disease (NAFLD).

[0002] Incorporation by Reference of Electronically Submitted Materials

[0003] Incorporated herein by reference in its entirety is the Nucleotide / Amino Acid Sequence Listing filed concurrently herewith and identified as follows: A 550 kilobyte XML file named "A-2911-WO01-SEC.xml" created on May 19, 2023.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 345,513, filed May 25, 2022, which is hereby incorporated by reference in its entirety. Background Art

[0006] Nonalcoholic fatty liver disease (NAFLD) constitutes a spectrum of liver pathologies and is the most common chronic liver disease in the world, with its prevalence doubling in the past 20 years and now 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 as the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes in individuals who 1) have no history of significant alcohol consumption and 2) in whom other types of liver disease have been excluded (Zhu et al. (2016) World J Gastroenterol 22(36):8226-33; Rinella (2015) JAMA 313(22):2263-73; Yki-Jarvinen (2016) Diabetologia 59(6):1104-11). In some individuals, the accumulation of ectopic fat in the liver (called steatosis) triggers inflammation and hepatocellular damage, leading to a more advanced disease called nonalcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, an estimated 75 to 100 million Americans have NAFLD, with NASH accounting for approximately 10%-30% of NAFLD diagnoses (Rinella, supra; Younossi et al. (2016) Hepatology 64(5):1577-1586).

[0007] One of the key triggers for the onset of NAFLD is the loss of triglyceride (TG) homeostasis in the liver, which leads to increased accumulation of TG and fatty acids. Over time, lipotoxicity exhausts the adaptive and regenerative responses of hepatocytes, leading to inflammation, activation of the innate immune system, and fatty liver inflammation. Progressive NASH triggers collagen deposition or fibrosis in the liver. As the grade of fibrosis worsens, the liver of 10%-29% of NASH individuals becomes cirrhotic and eventually leads to hepatocellular carcinoma.

[0008] The sterol response element binding protein (SREBP) family plays an important role in the regulation of de novo lipogenesis and TG accumulation in the liver. In the endoplasmic reticulum (ER), SREBP is synthesized in the form of an inactive precursor. SREBP cleavage activation protein (SCAP) is the only known regulator of transcription factors of the SREBP family. Immediately after synthesis, SCAP forms a complex with SREBP and escorts SREBP to the Golgi vesicle. Here, SREBP is further processed to release the active amino terminus of the transcription factor. Active SREBP translocates to the nucleus and binds to the SREBP response element to drive transcriptional activation of target genes. Therefore, inhibiting SCAP function can prevent the processing of active SREBP and reduce lipogenesis and TG accumulation in the liver.

[0009] Currently, NAFLD symptoms are managed via weight loss and treatment of any secondary conditions, as there are no approved pharmacological treatments.Therefore, there is a need for compositions and methods for treating NAFLD in affected individuals. Summary of the invention

[0010] The present disclosure provides an RNAi construct comprising a sense strand and an antisense strand, wherein the RNAi construct inhibits the expression of SREBP cleavage activation protein (SCAP) mRNA. In certain embodiments, the RNAi construct comprises a region of at least 15 consecutive nucleotides having no more than 3 nucleotide differences from the antisense sequences listed in Table 1.

[0011] In some embodiments, the sense strand of the RNAi construct described herein comprises a sequence that is fully complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, the length of the sense strand and the antisense strand is about 15 to about 30 nucleotides respectively. In some embodiments, the RNAi construct comprises at least one blunt end. In other embodiments, the RNAi construct comprises at least one nucleotide overhang. Such a nucleotide overhang may comprise at least 1 to 6 unpaired nucleotides and may be located at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' end of both the sense strand and the antisense strand. In certain embodiments, the RNAi construct comprises an overhang of two unpaired nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct comprises an overhang with two unpaired nucleotides at the 3' end of the antisense strand, and comprises a blunt end at the 3' end of the sense strand / 5' end of the antisense strand.

[0012] The RNAi construct of the present disclosure may include one or more modified nucleotides, including nucleotides with modifications to the ribose ring, core base or phosphodiester backbone. In certain embodiments, the modification of the ribose ring of the RNAi construct includes one or more 2'-modifications. Such 2'-modifications may include nucleotides modified by 2'-fluorine, nucleotides modified by 2'-O-methyl, nucleotides modified by 2'-O-methoxyethyl, nucleotides modified by 2'-O-allyl or bicyclic nucleic acids (BNA). The modification of the ribose ring may also include the incorporation of glycol nucleic acids (GNA), wherein the ribose ring is replaced by propylene glycol. In a specific embodiment, the RNAi construct includes one or more nucleotides modified by 2'-fluorine, nucleotides modified by 2'-O-methyl, or a combination thereof. In certain embodiments, all nucleotides in the sense strand and antisense strand of the RNAi construct are modified nucleotides. Abasic nucleotides may be incorporated into the RNAi construct of the present disclosure, for example, as the terminal nucleotides of the 3' end, 5' end, or both 3' end and 5' end of the sense strand. In such embodiments, the abasic nucleotide may be inverted, for example linked to an adjacent nucleotide by a 3'-3' internucleotide bond or a 5'-5' internucleotide bond.

[0013] In some embodiments, the RNAi construct comprises at least one backbone modification, such as a modified internucleotide or internucleoside bond. In certain embodiments, the RNAi construct described herein comprises at least one phosphorothioate internucleotide bond. In particular embodiments, the phosphorothioate internucleotide bond may be located at the 3' or 5' end of the sense strand and / or antisense strand.

[0014] The present disclosure also provides a composition comprising the above RNAi construct and a pharmaceutically acceptable carrier, excipient or diluent; and a method for reducing SCAP expression in a patient in need thereof, the methods comprising administering the above RNAi construct or composition to the patient.

[0015] The present disclosure also includes RNAi constructs for reducing SCAP expression, such as for treating NAFLD or NASH. Also provided is the use of RNAi constructs that inhibit SCAP expression for preparing a medicament for treating NAFLD. DETAILED DESCRIPTION

[0016] The present disclosure is based in part on the design and generation of RNAi constructs that target the SREBP cleavage activating protein (SCAP) gene and reduce the expression of SCAP in liver cells. Inhibition of SCAP expression can be used to treat or prevent conditions associated with SCAP expression, including liver-related diseases such as simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible advanced liver scarring), or SCAP-mediated hyperlipidemia or hypertriglyceridemia.

[0017] The present disclosure provides compositions and methods for regulating the expression of genes encoding SCAP. In some embodiments, the gene may be in a cell or subject, such as a mammal (e.g., a human). In some embodiments, the compositions of the present disclosure include an RNAi construct that targets SCAP mRNA and reduces the expression of SCAP in a cell or mammal. Such RNAi constructs can be used to treat or prevent various forms of liver-related diseases, such as simple fatty liver (steatosis), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible late-stage liver scarring), or SCAP-mediated hyperlipidemia or hypertriglyceridemia (see, e.g., Lee et al., Experimental & Molecular Medicine, 52: 724-729 (2020)).

[0018] As discussed above, SCAP (SREBP cleavage activation protein) is a sterol-regulated escort protein that transports SREBP from its synthesis site in the endoplasmic reticulum to its cleavage site in the Golgi apparatus, where they are sequentially cleaved by two proteases that release the cytoplasmic NH2-terminal transcription factor domain. Cell culture experiments have shown 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 cannot synthesize cholesterol, and they require an external source of cholesterol to grow (Rawson et al., J Biol Chem. [Journal of Biological Chemistry], 274: 28549-28556 (1999)). SCAP also acts as a cholesterol sensor, which is mediated by the multi-origin membrane domain of SCAP. Point mutations in this sterol sensing motif prevent sterol containment of SREBP cleavage and lead to unregulated overproduction of cholesterol.

[0019] The control of lipid synthesis is particularly important in the liver, which synthesizes lipids not only for its own use but also for export to the plasma as lipoproteins. The level of plasma lipoprotein cholesterol is reduced by treatment with statins. In addition, when plasma insulin is elevated (as may occur in obesity and non-insulin-dependent diabetes), fatty acid synthesis by the liver is elevated. In the liver, overexpression of the soluble transcriptional activation domain of SREBP-1a in transgenic mice leads to large fatty livers. Transgenic mice that overexpress the sterol-resistant mutant SCAP develop excessive cholesterol production in the liver and fail to exhibit normal feedback inhibition when fed cholesterol, indicating that SCAP plays a regulatory role in the liver. Mice with conditional SCAP deficiency in the liver exhibit reduced basal rates of cholesterol and fatty acid synthesis in the liver, primarily due to a reduction in mRNA encoding multiple biosynthetic enzymes (Matsuda et al., Genes & Development 15:1206-121 (2001)). In addition, NASH / NAFLD patients exhibit increased expression and transcriptional activity of SREBP1c and its target genes. Taken together, these data provide in vivo evidence that SCAP and SREBP are required for hepatic lipid synthesis.

[0020] RNA interference (RNAi) is a process in which exogenous RNA is introduced into cells, resulting in specific degradation of mRNA encoding a targeted protein, leading to reduced protein expression. Advances in both RNAi technology and liver delivery and increasing positive results with other RNAi-based therapies indicate that RNAi is an effective means of therapeutically treating NAFLD by directly targeting genes that regulate lipid synthesis in the liver, such as SCAP.

[0021] As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of a target gene (e.g., SCAP) via an RNA interference mechanism. "RNA interference" is a process in which a nucleic acid molecule induces the cutting and degradation of a target RNA molecule (e.g., messenger RNA or mRNA molecule) in a sequence-specific manner (e.g., by an RNA-induced silencing complex (RISC) pathway). In some embodiments, an RNAi construct comprises a double-stranded RNA (dsRNA) molecule comprising two antiparallel continuous nucleotide chains that are fully complementary to each other to hybridize to form a duplex region. A double-stranded RNAi construct may also be referred to as an RNAi "trigger." The term "hybridize or hybridization" refers to the pairing of complementary polynucleotides, typically via hydrogen bonding (e.g., Watson-Crick hydrogen bonding, Hoogsteen hydrogen bonding, or reverse Hoogsteen hydrogen bonding) between complementary bases in two polynucleotides. A chain comprising a region having a sequence that is substantially complementary to a target sequence (e.g., a target mRNA) is referred to as an "antisense strand." "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.

[0022] In certain embodiments, the sense strand and antisense strand of double-stranded RNA can be two separate molecules, which hybridize to form a duplex region, otherwise they are unconnected. Such double-stranded RNA molecules formed by two separate chains are called "small interfering RNA" or "short interfering RNA" (siRNA). siRNA is a class of non-coding double-stranded RNA molecules, which are typically about 20-27 base pairs and are the core of RNAi. Therefore, in some embodiments, the RNAi construct of the present disclosure includes siRNA. In other embodiments, the RNAi construct can be microRNA (also referred to as "miRNA" or "mature miRNA"). MiRNA is a small (about 18-24 nucleotides in length) non-coding RNA molecule present in plants, animals and some viruses. MiRNA is similar to siRNA, but miRNA originates from endogenous precursor hairpin RNA structure. MiRNA regulates gene expression by base pairing with the complementary region of target mRNA and guiding the cutting of target RNA via RISC pathway.

[0023] In some embodiments, the present disclosure provides an RNAi construct for SCAP. In some embodiments, the RNAi construct is a siRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a SCAP mRNA sequence. The region of the RNAi antisense strand can be complementary to any suitable region of the SCAP mRNA sequence. For example, the antisense strand can comprise a region complementary to a coding region or a 3' untranslated region (UTR) of a SCAP mRNA sequence.

[0024] Double-stranded RNAi molecules can include chemical modifications to ribonucleotides, including modifications to the ribose, base or backbone components of ribonucleotides, as described herein or known in the art. For the purposes of this disclosure, the term "double-stranded RNA" encompasses any such modifications as employed in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.).

[0025] As used herein, if a polynucleotide comprising a first sequence can hybridize with a polynucleotide comprising a second sequence to form a duplex region under certain conditions (such as physiological conditions), the first sequence is "complementary" to the second sequence. Other such conditions may include moderate or stringent hybridization conditions known to those of ordinary skill in the art. If a polynucleotide comprising a first sequence is base-paired with a polynucleotide comprising a second sequence over the entire length of one or both nucleotide sequences without any mismatches, the first sequence is considered to be completely complementary to the second sequence (100% complementary). If a sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to a target sequence, the sequence is "substantially 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. If no more than 5, 4, 3, 2 or 1 mismatches are present in a duplex region of 30 base pairs when the two sequences hybridize, it can also be said that the sequence is substantially complementary to another sequence. Generally, if any nucleotide overhangs as defined herein are present, the sequence of such overhangs is not considered when determining the degree of complementarity between two sequences. For example, a sense strand of 21 nucleotides in length and an antisense strand of 21 nucleotides in length hybridizing to form a 19 base pair duplex region with a 2 nucleotide overhang at the 3' end of each strand would be considered to be fully complementary as that term is used herein.

[0026] In some embodiments, the region of the antisense strand comprises a sequence that is completely complementary to a region of a target RNA sequence (e.g., SCAP mRNA). In such embodiments, the sense strand may comprise a sequence that is completely complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, such as 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense strand and the antisense strand. In certain embodiments, any mismatch preferably occurs in the terminal region (e.g., within 6, 5, 4, 3, 2, or 1 nucleotides of the 5' end and / or 3' end of the strand). In one embodiment, any mismatch in the duplex region formed by the sense strand and the antisense strand ideally occurs within 6, 5, 4, 3, 2, or 1 nucleotides of the 5' end of the antisense strand.

[0027] When the two substantially complementary chains of dsRNA are composed of separate RNA molecules, those molecules do not need to be but can be covalently linked. If the two chains are covalently linked to form a duplex structure in a manner other than an uninterrupted nucleotide chain between the 3' end of one chain and the 5' end of the corresponding other chain, the connecting structure is referred to as a "joint". The RNA chains may have the same or different numbers of nucleotides. The maximum number of base pairs in the duplex is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi may include one or more nucleotide overhangs.

[0028] In other embodiments, the sense strand and antisense strand that hybridize to form a duplex region can be part of a single RNA molecule, that is, the sense strand and antisense strand are part of the self-complementary region of a single RNA molecule. In such a case, a single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by connecting the continuous unpaired nucleotide sequence forming the loop region to the 5' end of the antisense strand. The loop region typically has a sufficient length to allow the RNA molecule to fold back on itself so that the antisense strand can be base-paired with the sense strand to form a duplex or stem region. The loop region can include about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. As noted herein, such RNA molecules with at least a partial self-complementary region are referred to as "short hairpin RNA" (shRNA). In some embodiments, the loop region may include at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In other embodiments, the loop region may have 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer unpaired nucleotides. In certain embodiments, the RNAi construct disclosed herein comprises shRNA. The length of a single at least partially self-complementary RNA molecule can be about 35 nucleotides to about 100 nucleotides, about 45 nucleotides to about 85 nucleotides, or about 50 to about 60 nucleotides, and comprises a duplex region and a loop region, each region having a length recited herein.

[0029] In some embodiments, the RNAi construct disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence that is substantially or completely complementary to a SCAP messenger RNA (mRNA) sequence. As used herein, a "SCAP mRNA sequence" refers to any messenger RNA sequence encoding a SCAP protein, including a SCAP protein variant or isoform from any species (e.g., mouse, rat, non-human primate, human), including splice variants. SCAP is also known in the art as a SREBF partner.

[0030] SCAP mRNA sequences also include transcript sequences expressed as their complementary DNA (cDNA) sequences. A cDNA sequence refers to a sequence of an mRNA transcript expressed as DNA bases (e.g., guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil, and cytosine). Thus, the antisense strand of the RNAi construct disclosed herein may include a region having a sequence that is substantially or completely complementary to a target SCAP mRNA sequence or a SCAP cDNA sequence. The SCAP mRNA or cDNA sequence may include, but is not limited to, any SCAP mRNA or cDNA sequence, such as may be derived from NCBI reference sequences NM_001320044.2 or NM_012235.4.

[0031] The region of the antisense strand may be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the SCAP mRNA sequence. In some embodiments, the target region of the SCAP mRNA sequence to which the antisense strand comprises a region complementary thereto may range from about 15 to about 30 consecutive nucleotides, from about 16 to about 28 consecutive nucleotides, from about 18 to about 26 consecutive nucleotides, from about 17 to about 24 consecutive nucleotides, from about 19 to about 25 consecutive nucleotides, from about 19 to about 23 (e.g., 19, 20, 21, 22, or 23) consecutive nucleotides, or from about 19 to about 21 consecutive nucleotides. In certain embodiments, the region of the antisense strand comprising a sequence that is substantially or fully complementary to the SCAP mRNA sequence may, in some embodiments, comprise at least 19 consecutive nucleotides from the antisense sequences listed in Table 1. In some embodiments, the sense and / or antisense sequence comprises at least 15 consecutive nucleotides (e.g., at least 16, 17, or 18 consecutive nucleotides) from the sequences listed in Table 1 with no more than 1, 2, or 3 nucleotide mismatches.

[0032] The sense strand of RNAi constructs typically comprises a sequence fully complementary to the antisense strand so that the two chains hybridize under physiological conditions to form a sequence in a duplex region. "Duplex region" refers to a region in two complementary or substantially complementary polynucleotides, which interact to form base pairs with each other through Watson-Crick base pairing or other hydrogen bonding, thereby producing a duplex between the two polynucleotides. The duplex region of RNAi constructs should have enough length to allow RNAi constructs to enter RNA interference pathways, for example, by engaging Dicer enzymes and / or RISC complexes (described below). For example, in some embodiments, the length of the duplex region is about 15 to about 30 base pairs. Other lengths of the duplex region within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the length of the duplex region is about 17 to about 24 base pairs. In another embodiment, the length of the duplex region is about 19 to about 21 base pairs. For example, the length of the duplex region can be about 19 base pairs.

[0033] In some embodiments, the RNAi constructs disclosed herein contain a duplex region of about 17 to about 24 nucleotides that interacts with a target RNA sequence (e.g., a SCAP target mRNA sequence) to guide the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells can be broken down into siRNAs by a type III endonuclease called Dicer (Sharp et al. (2001) Genes Dev. [Genes and Development] 15: 485). Dicer is a ribonuclease-III-like enzyme that processes dsRNA into 19-23 base pair short interfering RNAs with a characteristic two base 3' overhang (Bernstein et al., (2001) Nature [Nature] 409: 363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell [Cell], 107: 309). After binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0034] For embodiments where the sense strand and the antisense strand are two separate molecules (e.g., siRNA RNAi constructs), the length of the sense strand and the antisense strand need not be the same as the length of the duplex region. For example, one or both strands may be longer than the duplex region and have one or more unpaired nucleotides or mismatches flanking the duplex region. Therefore, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides extending beyond the duplex region at the end of the strand. When the 3' end of a strand extends beyond the 5' end of another strand or when the 5' end of a strand extends beyond the 3' end of another strand, a nucleotide overhang is typically produced. The length of the nucleotide overhang is typically between 1 and 6 nucleotides, between 1 and 5 nucleotides, between 1 and 4 nucleotides, between 1 and 3 nucleotides, between 2 and 6 nucleotides, between 2 and 5 nucleotides, or between 2 and 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5 or 6 nucleotides. In a specific embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang may be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some embodiments, the overhang comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide may comprise a ribonucleotide or a modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the overhang comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide.

[0035] The nucleotide overhangs may be at the 5' end or 3' end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5' end and 3' end of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5' end and 3' end of the sense strand. In some embodiments, the RNAi construct comprises nucleotide overhangs at the 5' end of the sense strand and the 5' end of the antisense strand. In other embodiments, the RNAi construct comprises nucleotide overhangs at the 3' end of the sense strand and the 3' end of the antisense strand.

[0036] RNAi constructs can include a single nucleotide overhang at one end of a double-stranded RNA molecule, and a blunt end at the other end. "Blunt end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and there are no unpaired nucleotides extending beyond the duplex region. In certain embodiments, the RNAi construct includes a nucleotide overhang at the 3' end of the sense strand, and includes a blunt end 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 includes a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct includes a blunt end at both ends of the double-stranded RNA molecule. In such an embodiment, the sense strand and the antisense strand have the same length, and the length of the duplex region is the same as the sense strand and the antisense strand (that is, the molecule is double-stranded over its entire length).

[0037] The sense strand and the antisense strand can each independently be any suitable length, such as about 15 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and the antisense strand are each about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and the antisense strand have the same length, but form a duplex region that is shorter than these strands so that the RNAi construct has a two nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each having a length of 21 nucleotides, (ii) a duplex region of 19 base pairs in length, and (iii) a nucleotide overhang of 2 unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each having a length of 23 nucleotides, (ii) a duplex region of 21 base pairs in length, and (iii) a nucleotide overhang of 2 unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the sense strand and the antisense strand have the same length and form a duplex region over their entire length, so that there is no nucleotide overhang at either end of the double-stranded molecule. In one such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand each having a length of 21 nucleotides and (ii) a duplex region of 21 base pairs in length. In another embodiment, the RNAi construct is blunt ended and comprises (i) a sense strand and an antisense strand each of 23 nucleotides in length and (ii) a duplex region of 23 base pairs in length.

[0038] In other embodiments, the sense strand or the antisense strand is longer than the other strand, and the two strands form a duplex region with a length equal to the shorter strand length so that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand of 19 nucleotides in length, (ii) an antisense strand of 21 nucleotides in length, (iii) a duplex region of 19 base pairs in length, and (iv) a single nucleotide overhang with 2 unpaired nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand of 21 nucleotides in length, (ii) an antisense strand of 23 nucleotides in length, (iii) a duplex region of 21 base pairs in length, and (iv) a single nucleotide overhang with 2 unpaired nucleotides at the 3' end of the antisense strand.

[0039] The antisense strand of the RNAi construct disclosed herein may comprise 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 antisense sequence listed in Table 1 comprises a sequence of 16-19 consecutive nucleotides complementary to the SCAP mRNA sequence plus a two-nucleotide overhang sequence. Thus, in some embodiments, the antisense strand comprises 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 may comprise 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 comprises a 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.

[0040] Modified nucleotides

[0041] The RNAi construct disclosed herein (such as those listed in Table 1) can include one or more modified nucleotides. "Modified nucleotides" refer to nucleotides with one or more chemical modifications to nucleosides, core bases, pentose rings, or phosphate groups. As used herein, modified nucleotides do not encompass ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate and cytidine monophosphate, and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. However, RNAi constructs can include a combination of modified nucleotides, ribonucleotides and deoxyribonucleotides. Modified nucleotides are incorporated into one or two chains of double-stranded RNA molecules, and the in vivo stability of RNA molecules can be improved, for example, by reducing the sensitivity of molecules to nucleases and other degradation processes. The effectiveness of RNAi constructs to reduce target gene expression can also be enhanced by incorporating modified nucleotides.

[0042] In certain embodiments, the modified nucleotide has the modification of ribose. These sugar modifications may include modifications at the 2' and / or 5' positions of the pentose ring and bicyclic sugar modifications. The 2'-modified nucleotide refers to a nucleotide with a pentose ring, which has a substituent other than H or OH at the 2' position. Such 2'-modifications include but are not limited to 2'-O-alkyl (e.g., O-C1-C10 or O-C1-C10 substituted alkyl), 2'-O-allyl (O-CH2CH=CH2), 2'-C-allyl, 2'-fluorine, 2'-O-methyl (OCH3), 2'-O-methoxyethyl (O-(CH2)2OCH3), 2'-OCF3, 2'-O(CH2)2SCH3, 2'-O-aminoalkyl, 2'-amino (e.g., NH2), 2'-O-ethylamine and 2'-azido. Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy.

[0043] "Bicyclic sugar modification" refers to the modification of a pentose ring, wherein a bridge connects two atoms of the ring to form a second ring to produce a bicyclic sugar structure. In certain embodiments, the bicyclic sugar modification is included in the bridge between the 4' and 2' carbons of the pentose ring. The nucleotides comprising the sugar moieties with bicyclic sugar modifications are referred to herein as "bicyclic nucleic acids", "bridged nucleic acids" or "BNAs". "Locked nucleic acids" (LNA) are 2', 4'-bicyclic nucleic acids (2', 4'-BNAs), wherein the ribose ring is locked by a methylene bridge connecting 2'-oxygen and 4'-carbon. Exemplary bicyclic sugar modifications include, but are not limited to, α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleic acids (BNA); β-D-methyleneoxy (4'-CH2-O-2') BNA (LNA); ethyleneoxy (4'-(CH2)2-O-2') BNA; aminooxy (4'-CH2-ON(R)-2') BNA; oxyamino (4'-CH2-N(R)-O-2') BNA; methyl(methyleneoxy) (4'-CH(CH3 )-O-2') BNA (also known as constrained ethyl or cEt); methylene-thio (4'-CH2-S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methyl carbocycle (4'-CH2-CH(CH3)-2') BNA; propylene carbocycle (4'-(CH2)3-2') BNA; and methoxy (ethyleneoxy) (4'-CH(CH2OMe)-O-2') BNA (also known 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. Pat. No. 9,181,551, U.S. Pat. Pub. No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, 19:937-954 (2012).

[0044] In some embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In a specific embodiment, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0045] Both the sense strand and the antisense strand of the RNAi construct may comprise 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 in the sense strand are modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides may be 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or a combination thereof.

[0046] In certain embodiments, the modified nucleotides incorporated into one or both strands of the RNAi constructs of the invention have modifications of nucleobases (also referred to herein as "bases"). "Modified nucleobases" or "modified bases" refer to bases other than the naturally occurring purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). The modified nucleobases may be synthetic or naturally occurring modifications and include, but are not limited to, universal bases, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and Cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo (especially 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.

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

[0048] Other suitable modified bases that can be incorporated into the RNAi 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 hereby incorporated by reference in their entirety. It is well known to those skilled in the art that guanine, cytosine, adenine, thymine and uracil can be replaced by other nucleobases (such as the modified nucleobases described above) without substantially changing the base pairing properties of the polynucleotide comprising nucleotides carrying such substituted nucleobases.

[0049] In certain embodiments, the sense strand and antisense strand of RNAi constructs may include one or more abasic nucleotides."Abasic nucleotides" or "abasic nucleosides" are nucleotides or nucleosides lacking a nucleobase at the 1' position of ribose. In certain embodiments, abasic nucleotides are incorporated into the ends of the sense strand and / or antisense strand of RNAi constructs. In one embodiment, the sense strand includes abasic nucleotides as terminal nucleotides at its 3' end, its 5' end, or its 3' and 5' ends. In another embodiment, the antisense strand includes abasic nucleotides as terminal nucleotides at its 3' end, its 5' end, or its 3' and 5' ends. In such embodiments where abasic nucleotides are terminal nucleotides, they may be reverse nucleotides, i.e., connected to adjacent nucleotides by 3'-3' internucleotide bonds (when at the 3' end of the chain) or 5'-5' internucleotide bonds (when at the 5' end of the chain) rather than natural 3'-5' internucleotide bonds. Abasic nucleotides may also include sugar modifications, such as any of the above-mentioned sugar modifications. In certain embodiments, the abasic nucleotide comprises 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 comprises a 2'-O-methyl modification. In another embodiment, the abasic nucleotide comprises a 2'-H modification (i.e., a deoxy abasic nucleotide).

[0050] In some embodiments, all pyrimidine nucleotides preceding the adenosine nucleotide in the sense strand and / or antisense strand are modified nucleotides. For example, when the sequence 5'-CA-3' or 5'-UA-3' occurs in either strand, the cytidine and uridine nucleotides are modified nucleotides, preferably 2'-O-methyl modified nucleotides. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides), and the 5' nucleotides in all occurrences of the sequence 5'-CA-3' or 5'-UA-3' in the antisense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides). In other embodiments, all nucleotides in the duplex region are modified nucleotides. In such embodiments, the modified nucleotides are preferably 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

[0051] In embodiments where the RNAi construct comprises a nucleotide overhang, the nucleotide in the overhang may be a ribonucleotide, a deoxyribonucleotide, or a modified nucleotide. In one embodiment, the nucleotide in the overhang is a deoxyribonucleotide, such as deoxythymidine. In another embodiment, the nucleotide in the overhang is a modified nucleotide. For example, in some embodiments, the nucleotide in the overhang is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-methoxyethyl modified nucleotide, or a combination thereof.

[0052] The RNAi construct of the present disclosure can also include one or more modified internucleotide bonds. As used herein, the term "modified internucleotide bond" refers to a bond between nucleotides except a natural 3' to 5' phosphodiester bond. In certain embodiments, the modified internucleotide bond is a phosphorus-containing internucleotide bond, such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a thiophosphate (P=S), a chiral thiophosphate, a dithiophosphate, a thiophosphoramidate, an alkylthiophosphonate, an alkylthiophosphotriester, and a boranephosphate. In one embodiment, the modified internucleotide bond is a 2' to 5' phosphodiester bond. In other embodiments, the modified internucleotide bond is a phosphorus-free internucleotide bond, and therefore can be referred to as a modified internucleoside bond. Such phosphorus-free bonds include, but are not limited to, morpholine bonds (formed in part by the sugar portion of the nucleoside); siloxane bonds (-O-Si(H)2-O-); sulfide, sulfoxide and sulfone bonds; formyl and thioformyl bonds; olefin-containing backbones; aminosulfonate backbones; methylenemethylimino (-CH2-N(CH3)-O-CH2-) and methylenehydrazine bonds; sulfonate and sulfonamide bonds; amide bonds; and other bonds with mixed N, O, S and CH2 component parts. In one embodiment, the modified internucleoside bond is a peptide-based bond (e.g., aminoethylglycine) that produces peptide nucleic acids or PNAs, such as those described in U.S. Pat. Nos. 5,539,082, 5,714,331 and 5,719,262. Other suitable modified internucleotide and internucleoside linkages that can be used in the disclosed RNAi constructs are described in U.S. Patents 6,693,187 and 9,181,551, U.S. Patent Publication No. 2016 / 0122761, and Deleavey and Damha, supra.

[0053] In certain embodiments, the RNAi construct comprises one or more phosphorothioate internucleotide bonds. The phosphorothioate internucleotide bonds can be present in the sense strand, antisense strand or two chains 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 bonds. In other embodiments, the antisense strand comprises 1,2,3,4,5,6,7,8 or more phosphorothioate internucleotide bonds. In still other embodiments, the two chains comprise 1,2,3,4,5,6,7,8 or more phosphorothioate internucleotide bonds. The RNAi construct can comprise one or more phosphorothioate internucleotide bonds at the 3'-end, 5'-end or 3'-end and 5'-end of the sense strand, antisense strand or two chains. 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) continuous phosphorothioate nucleotide inter-bonds at the 3' end of the sense strand, antisense strand or two chains. 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) continuous phosphorothioate nucleotide inter-bonds at the 5' end of the sense strand, antisense strand or two chains. In one embodiment, the RNAi construct is included in a single phosphorothioate nucleotide inter-bond at the 3' end of the sense strand and a single phosphorothioate nucleotide inter-bond at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises two continuous phosphorothioate nucleotide inter-bonds (i.e., the phosphorothioate nucleotide inter-bond at the first and second nucleotides at the 3' end of the antisense strand) at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises two continuous phosphorothioate nucleotide inter-bonds at the 3' end and 5' end of the antisense strand. In another embodiment, the RNAi construct comprises two continuous phosphorothioate internucleotide bonds at the 3' end and 5' end of the antisense strand, and comprises two continuous phosphorothioate internucleotide bonds at the 5' end of the sense strand. In still another embodiment, the RNAi construct comprises two continuous phosphorothioate internucleotide bonds at the 3' end and 5' end of the antisense strand, and comprises two continuous phosphorothioate internucleotide bonds at the 3' end and 5' end of the sense strand (i.e., the phosphorothioate internucleotide bonds at the first and second internucleotide bonds at the 5' end and 3' end of the antisense strand and the phosphorothioate internucleotide bonds at the first and second internucleotide bonds at the 5' end and 3' end of the sense strand). In any embodiment in which one or both chains comprise one or more phosphorothioate internucleotide bonds, the remaining internucleotide bonds in the chain may be natural 3' to 5' phosphodiester bonds. For example, in some embodiments, each internucleotide bond in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, wherein at least one internucleotide bond is phosphorothioate.

[0054] In the embodiment that RNAi construct comprises nucleotide overhang, two or more unpaired nucleotides in this overhang can be connected by key between thiophosphate nucleotide.In certain embodiments, all unpaired nucleotides in the nucleotide overhang at 3 ' end of antisense strand and / or sense strand are connected by key between thiophosphate nucleotide.In other embodiments, all unpaired nucleotides in the nucleotide overhang at 5 ' end of antisense strand and / or sense strand are connected by key between thiophosphate nucleotide.In still other embodiments, all unpaired nucleotides in any nucleotide overhang are connected by key between thiophosphate nucleotide.

[0055] In some embodiments, the 5' end of the sense strand, the antisense strand, or both the antisense strand and the sense strand of the disclosed RNAi construct comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to a terminal phosphate group including unmodified phosphates (-OP=O)(OH)OH) and modified phosphates. Modified phosphates include phosphates in which one or more of the O and OH groups are replaced by H, O, S, N(R) or alkyl, wherein R is H, an amino protecting group, or an unsubstituted or substituted alkyl. Exemplary phosphate moieties include, but are not limited to, 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or non-methylated); 5'-adenosine cap or any other modified or unmodified nucleotide cap structure; 5'-monothioate (phosphorothioate); 5'-monodithioate (phosphorodithioate); 5'-α-thiotriphosphate; 5'-γ-thiotriphosphate, 5'-phosphoramidate; 5'-vinyl phosphate; 5'-alkylphosphonates (wherein "alkyl" can be methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (wherein "alkyl ether" can be methoxymethyl, ethoxymethyl, etc.).

[0056] The modified nucleotides that can be incorporated into the RNAi constructs disclosed herein can have more than one chemical modification described herein. For example, the modified nucleotides can have modifications to ribose and modifications to the nucleobase. For example, the modified nucleotides can include 2' sugar modifications (e.g., 2'-fluoro or 2'-O-methyl) and include modified bases (e.g., 5-methylcytosine or pseudouracil). In other embodiments, the modified nucleotides can include a combination of sugar modifications and modifications to 5' phosphate, and when the modified nucleotides are incorporated into polynucleotides, these modifications will produce modified internucleotides or internucleoside bonds. For example, in some embodiments, the modified nucleotides can include sugar modifications, such as 2'-fluoro modifications, 2'-O-methyl modifications, or bicyclic sugar modifications, and 5' thiophosphate groups. Therefore, in some embodiments, one or both chains of the RNAi constructs include a combination of 2' modified nucleotides or BNAs and thiophosphate internucleotide bonds. In certain embodiments, both the sense and antisense strands of the RNAi constructs comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide bonds. Exemplary RNAi constructs comprising modified nucleotides and internucleotide bonds are shown in Tables 1 and 2.

[0057] Function of RNAi constructs

[0058] The RNAi constructs disclosed herein desirably reduce or inhibit the expression of SCAP in cells, particularly liver cells. Thus, in one embodiment, the disclosure provides a method for reducing the expression of SCAP in a cell by contacting the cell with any of the RNAi constructs described herein. The cell can be in vitro or in vivo. SCAP expression can be assessed by measuring the amount or level of SCAP mRNA, SCAP protein, or another biomarker associated with SCAP expression. Reduction in SCAP expression in cells or animals treated with the RNAi constructs disclosed herein can be determined relative to SCAP expression in cells or animals not treated with the RNAi construct or with a control RNAi construct. For example, in some embodiments, the reduction of SCAP expression is assessed 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 sequence or a scrambled sequence) or not treated with the construct; and (c) comparing the measured SCAP mRNA level from the treated cells in (a) with the measured SCAP mRNA level from the control cells in (b). Prior to comparison, the SCAP mRNA levels in the treated cells and the control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA). The SCAP mRNA level can be measured by a variety of methods, including northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

[0059] In other embodiments, the reduction of SCAP expression is assessed 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 with a nonsense sequence or a scrambled sequence) or not treated with the construct; and (c) comparing the measured SCAP protein level from the treated cells in (a) with the measured SCAP protein level from the control cells in (b). SCAP protein levels can be measured using any suitable method known to those skilled in the art, including, but not limited to, Western blot, immunoassay (e.g., ELISA), and flow cytometry. Any suitable method for measuring SCAP mRNA or protein can be used to assess the efficacy of the RNAi construct disclosed herein.

[0060] In some embodiments, the method of assessing the level of SCAP expression is performed in vitro in cells that naturally express SCAP (e.g., liver cells) or cells that have been engineered to express SCAP. In certain embodiments, these methods are performed in vitro in liver cells. Suitable liver cells include, but are not limited to, primary liver cells (e.g., liver cells of humans, non-human primates, or rodents), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the liver cell is a Hep3B cell. In another embodiment, the liver cell is a HepG2 cell.

[0061] In other embodiments, the method of assessing SCAP expression levels is performed in vivo. For example, the RNAi construct and any control RNAi construct can be administered to an animal (e.g., a rodent or non-human primate), and SCAP mRNA or protein levels can be assessed in liver tissue harvested from the animal after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with SCAP expression can be assessed in the treated animal.

[0062] In certain embodiments, the RNAi constructs disclosed herein reduce the expression of SCAP in liver cells by at least 40%, at least 45%, or at least 50%. In some embodiments, the RNAi constructs disclosed herein reduce the expression of SCAP in liver cells by at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%. In other embodiments, the RNAi constructs disclosed herein reduce the expression of SCAP in liver cells by about 90% or more, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. The percentage reduction in SCAP expression can be measured by any method described herein or other methods known in the art. For example, in certain embodiments, the RNAi constructs inhibit SCAP expression by at least 40% at 5 nM in vitro in Hep3B cells (containing wild-type SCAP). In related embodiments, the RNAi construct inhibits SCAP expression at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% at 5 nM in Hep3B cells in vitro. In other embodiments, the RNAi construct inhibits SCAP expression at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% at 5 nM in Hep3B cells in vitro. In certain embodiments, the RNAi construct disclosed herein inhibits SCAP expression at least 40% at 5 nM in HepG2 cells in vitro. In related embodiments, the RNAi construct disclosed herein inhibits SCAP expression at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% at 5 nM in HepG2 cells in vitro. In other embodiments, the RNAi construct inhibits SCAP expression at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% in HepG2 cells in vitro at 5 nM. In certain embodiments, the RNAi construct inhibits SCAP expression at least 40% in CHO transfected cells expressing human SCAP in vitro at 5 nM. In related embodiments, the RNAi construct inhibits SCAP expression at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in CHO transfected cells expressing human SCAP in vitro at 5 nM. In other embodiments, the RNAi construct inhibits SCAP expression at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% in CHO transfected cells expressing human SCAP in vitro at 5 nM.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).

[0063] In some embodiments, the IC is calculated 50 values ​​in order to assess the efficacy of the RNAi constructs disclosed herein in inhibiting the expression of SCAP in liver cells. 50 The "AC value" is the dose / concentration required to achieve 50% inhibition of biological or biochemical function. In other embodiments, the efficacy of the RNAi construct can be calculated 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 50 Values ​​can be determined by constructing dose-response curves and examining the effects of varying concentrations of the substance or antagonist on expression levels or functional activity in any assay. The IC value for a given antagonist or substance is 50 The IC value can be calculated by determining the concentration required to inhibit half of the maximal biological response or the native expression level. 50 The AC value can be calculated by determining the concentration of the RNAi construct required to inhibit the level of native SCAP expression in liver cells (e.g., the level of SCAP expression in control liver cells) by half in any assay (e.g., an immunoassay, an RNA FISH assay, or a droplet digital PCR assay). 50 The value can be calculated by determining the concentration required to activate half of the maximal biological response or the native expression level. The RNAi constructs disclosed herein can be expressed at an IC of less than about 20 nM (e.g., less than about 15 nM, 10 nM, 5 nM, or 1 nM). 50 Inhibit the expression of SCAP in hepatocytes (e.g., Hep3B cells). For example, the disclosed RNAi constructs can be expressed at 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 the expression of SCAP 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). 50Inhibit the expression of SCAP in liver cells (eg, Hep3B cells). The RNAi constructs disclosed herein can be used with an IC of less than about 20 nM. 50 Inhibit the expression of SCAP in liver cells (e.g., HepG2 cells). For example, the RNAi construct can be expressed at 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 the expression of SCAP 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 Inhibit the expression of SCAP in liver cells (eg, HepG2 cells). The RNAi constructs disclosed herein can be used with an IC of less than about 20 nM. 50 Inhibit the expression of SCAP in cells expressing human SCAP (e.g., CHO transfected 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 Inhibit the expression of SCAP in cells expressing SCAP. In certain embodiments, the RNAi construct has an IC of about 1 nM to about 10 nM (e.g., about 5 nM). 50 Inhibits the expression of SCAP in cells expressing SCAP.

[0064] The RNAi construct disclosed herein can be easily made using techniques known in the art (e.g., conventional nucleic acid solid phase synthesis). The polynucleotide of the RNAi construct can be assembled on a suitable nucleic acid synthesizer using standard nucleotides or nucleoside precursors (e.g., phosphoramidites). Automatic nucleic acid synthesizers are commercially sold by several suppliers, and these synthesizers include DNA / RNA synthesizers from Applied Biosystems (Applied Biosystems) (Foster City, California (Foster City, CA)), MerMade synthesizers from BioAutomation (Irving, Texas (Irving, TX)) and OligoPilot synthesizers from GE Healthcare Life Sciences (Pittsburgh, Pennsylvania (Pittsburgh, PA)).

[0065] 2' silyl protecting groups can be used in combination with the acid-labile dimethoxytrityl (DMT) at the 5' position of ribonucleosides to synthesize oligonucleotides via phosphoramidite chemistry. It is known that the final deprotection conditions will not significantly degrade the RNA product. All synthesis can be carried out on a large, medium or small scale in any automatic or manual synthesizer. Synthesis can also be carried out in multiwell plates, columns or slides.

[0066] 2'-O-silyl groups can be removed via exposure to fluoride ions, which can include any fluoride ion source, such as those salts containing fluoride ions paired with inorganic counterions (e.g., cesium fluoride and potassium fluoride) or those containing fluoride ions paired with organic counterions (e.g., tetraalkylammonium fluorides). Crown ether catalysts can be used in combination with inorganic fluorides in deprotection reactions. Exemplary fluoride ion sources include, but are not limited to, tetrabutylammonium fluoride or aminohydrofluorides (e.g., combining aqueous HF with triethylamine in a dipolar aprotic solvent (e.g., dimethylformamide)).

[0067] The choice of protecting groups on phosphite triesters and phosphate triesters can alter the stability of the triesters toward fluoride. Methyl protection of phosphate triesters or phosphite triesters can stabilize the linkage to fluoride ions and improve process yields.

[0068] Since ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in RNA with a protecting group orthogonal to the 5'-O-dimethoxytrityl protecting group (e.g., a protecting group that is stable to acid treatment). Silyl protecting groups meet this criterion and can be easily removed in the final fluoride deprotection step, which can result in minimal RNA degradation.

[0069] Tetrazolyl catalysts can be used in standard phosphoramidite coupling reactions. Exemplary catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylmercaptotetrazole, and p-nitrophenyltetrazole.

[0070] Additional methods of synthesizing the RNAi constructs described herein will be apparent to those of ordinary skill in the art.In addition, the various synthetic steps may be performed in an alternate sequence or order to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on bases), and protecting group methods (protection and deprotection) that can be used to synthesize the RNAi constructs described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and PGM Wuts, Protective Groups in Organic Synthesis, 2nd 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 (1996). Sons (1995) and subsequent versions. Custom synthesis of RNAi constructs is also available from several commercial suppliers, including Dharmacon, Inc. (Lafayette, CO), Axo Labs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0071] The RNAi construct disclosed herein may include a ligand. As used herein, a "ligand" refers to any compound or molecule that can interact directly or indirectly with another compound or molecule. The interaction of a ligand with another compound or molecule may trigger a biological response (e.g., initiate a signal transduction cascade, induce receptor-mediated endocytosis) or may be only physically associated. The ligand may modify one or more properties of the attached double-stranded RNA molecule, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties of the RNA molecule.

[0072] The ligand can comprise a serum protein (e.g., human serum albumin, low-density lipoprotein, globulin), a cholesterol moiety, a vitamin (e.g., biotin, vitamin E, vitamin B12), a folate moiety, a steroid, a bile acid (e.g., cholic acid), a fatty acid (e.g., palmitic acid, myristic acid), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), a glycoside, a phospholipid, or an antibody or binding fragment thereof (e.g., a whole antibody or binding fragment that targets an RNAi construct to a specific cell type, such as a liver cell). Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking 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., adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-di-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholic acid, dimethoxytrityl or phenoxazine), peptides (e.g., antenna peptides, Tat peptides, RGD peptides), alkylating agents, polymers (e.g., polyethylene glycol (PEG), PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0073] In certain embodiments, the ligand has endosomal lytic properties. The endosomal lytic ligand promotes endosomal lysis and / or RNAi constructs or components thereof from endosomes to the cytoplasm of cells. The endosomal lytic ligand can be a polycationic peptide or peptide mimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomal lytic ligand exhibits its active conformation at endosomal pH. An "active" conformation is one in which the endosomal lytic ligand promotes endosomal lysis and / or RNAi constructs or components thereof from endosomes to the cytoplasm of cells. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, Vol. 26: 2964-2972, 1987), EALA peptide (Vogel et al., J. Am. Chem. Soc., Vol. 118: 1581-1586, 1996), and their derivatives (Turk et al., Biochem. Biophys. Acta, Vol. 1559: 56-68, 2002). In one embodiment, the endosomolytic component may contain chemical groups (e.g., amino acids) that undergo changes in charge or protonation in response to changes in pH. The endosomolytic component may be linear or branched.

[0074] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroids. It is reported that the oligonucleotide conjugated with cholesterol is more active than its unconjugated counterpart (Manoharan, Antisense Nucleic Acid Drug Development [antisense nucleic acid drug development], Vol. 12: 103-228, 2002). The ligand comprising a cholesterol moiety and other lipids for conjugation with nucleic acid molecules has also been described in U.S. Patents 7,851,615, 7,745,608 and 7,833,992. In another embodiment, the ligand may comprise a folic acid moiety. The polynucleotide conjugated with the folic acid moiety can be taken up by cells via a receptor-mediated endocytosis pathway. Such folic acid-polynucleotide conjugates are described in, for example, U.S. Patent 8,188,247.

[0075] Considering that SCAP is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to deliver RNAi constructs specifically to liver cells. In certain embodiments, RNAi constructs can be specifically targeted to the liver by using ligands that bind or interact with proteins expressed on the surface of liver cells. For example, in certain embodiments, the ligands can include one or more antigen binding proteins (e.g., antibodies or binding fragments thereof (e.g., Fab, scFv)) that specifically bind to receptors expressed on liver cells.

[0076] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound consisting of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched or cyclic) and oxygen, nitrogen or sulfur atoms 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) and polysaccharides (e.g., starch, glycogen, cellulose and polysaccharide gum). In certain embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from pentose, hexose or heptose and a disaccharide and trisaccharide including such a monosaccharide unit. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine and N-acetylglucosamine.

[0077] In some embodiments, the ligand comprises a hexose or a hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine or N-acetyl-galactosamine. In a specific embodiment, the ligand comprises N-acetyl-galactosamine. The ligand comprising glucose, galactose and N-acetyl-galactosamine (GalNAc) is particularly effective in targeting compounds to liver cells (see, e.g., D'Souza and Devarajan, J.Control Release [Journal of Controlled Release], Vol. 203: 126-139, 2015). Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs disclosed herein are described in U.S. Patents 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Publication No. 2003 / 0130186; and WIPO Publication No. WO 2013 / 166155.

[0078] In certain embodiments, the ligand comprises a multivalent carbohydrate portion. As used herein, a "multivalent carbohydrate portion" refers to a portion comprising two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate portion comprises two or more binding domains consisting of carbohydrates, which can bind to two or more different molecules or two or more different sites on the same molecule. The valence of a carbohydrate portion represents the number of individual binding domains within the carbohydrate portion. For example, the terms "monovalent," "divalent," "trivalent," and "tetravalent" with respect to a carbohydrate portion refer to carbohydrate portions having one, two, three, and four binding domains, respectively. A multivalent carbohydrate portion may comprise a multivalent lactose portion, a multivalent galactose portion, a multivalent glucose portion, a multivalent N-acetyl-galactosamine portion, a multivalent N-acetyl-glucosamine portion, a multivalent mannose portion, or a multivalent fucose portion. In some embodiments, the ligand comprises a multivalent galactose portion. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine portion. In these and other embodiments, the multivalent carbohydrate moiety is divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety may be biantennary or triantennary. In a specific embodiment, the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another specific embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary GalNAc-containing ligands for incorporation into RNAi constructs disclosed herein include triantennary GalNAc-containing ligands (also referred to as "GalNAc3").

[0079] The ligand can be directly or indirectly attached or conjugated to the RNA molecule of the RNAi construct.For example, in some embodiments, the ligand is directly covalently attached to the sense strand or antisense strand of the RNAi construct.In other embodiments, the ligand is covalently attached to the sense strand or antisense strand of the RNAi construct via a joint.The ligand can be attached to the core base, sugar moiety or internucleotide bond of the polynucleotide (such as sense strand or antisense strand) of the RNAi construct disclosed herein.Conjugation or attachment with purine core base or its derivatives can occur at any position including endocyclic and exocyclic atoms.In certain embodiments, 2,6,7 or 8 positions of purine core base are attached to the ligand.Conjugation or attachment with pyrimidine core base or its derivatives can also occur at any position.In certain embodiments, 2,5 and 6 positions of pyrimidine core base can be attached to the ligand.Conjugation or attachment with the sugar moiety of nucleotide can occur at any carbon atom. The exemplary carbon atom that can be attached to the part of the sugar part includes 2', 3' and 5' carbon atoms. The 1' position can also be attached to the part, as in abasic nucleotides. The internucleotide bond can also support the attachment of the ligand. For phosphorus-containing bonds (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand can be directly attached to the phosphorus atom or to the O, N or S atom combined with the phosphorus atom. For the internucleoside bond (e.g., PNA) containing amines or amides, the ligand can be attached to the nitrogen atom of the amine or amide or to the adjacent carbon atom.

[0080] In certain embodiments, the ligand can be attached to the 3' end or 5' end of the sense strand or antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. For example, in some embodiments, the ligand is attached to the 5' terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 5' position of the 5' terminal nucleotide of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3' terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3' position of the 3' terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand, but 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.

[0081] In certain embodiments, the ligand is attached to the sense strand or antisense strand via a joint. A "joint" is an atom or group of atoms that covalently connects the ligand to the polynucleotide component of the RNAi construct. The joint can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In certain embodiments, the joint can include a bifunctional linking portion, which generally includes an alkyl portion with two functional groups. One of the functional groups is selected to bind to the compound of interest (e.g., the sense strand or antisense strand of the RNAi construct), and another is selected to substantially bind to any selected group, such as a ligand as described herein. In certain embodiments, the joint includes a chain structure or oligomer of a repeating unit (such as ethylene glycol or an amino acid unit). 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, the bifunctional linking moiety includes amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), etc.

[0082] Linkers that can be used to attach a ligand to the sense or antisense strand in an RNAi construct include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester, 6-aminohexanoic acid, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 alkenyl, or substituted or unsubstituted C2-C 10 Preferred substituent groups for such linkers include, but are not limited to, hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0083] In certain embodiments, the joint is cleavable. A cleavable joint is sufficiently stable outside the cell, but after entering the target cell, it is cut to release the joint of the two parts that the joint holds together. In certain embodiments, the cleavable joint is cut faster than in the subject's blood or under a second reference condition (which can be, for example, selected to simulate or represent conditions found in blood or serum) by at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 100 times in the target cell or under a first reference condition (which can, for example, be selected to simulate or represent conditions found in blood or serum).

[0084] Cleavable linkers are susceptible to cleavage agents (e.g., pH, redox potential, or the presence of degradable molecules). Typically, cleavage agents are more prevalent or found at higher levels or activity in cells than in serum or blood. Examples of such degradable agents include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidative or reductases or reductants (such as thiols) present in cells, which can degrade redox cleavable linkers by reduction; esterases; endosomes or agents that can form an acidic environment, such as those that produce a pH of five or less; enzymes, peptidases (which can be substrate-specific), and phosphatases that can hydrolyze or degrade acid-cleavable linkers by acting as a general acid.

[0085] The cleavable linker may comprise a portion susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, ranging from 5.5-6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand into the cell, or into a desired cellular compartment.

[0086] The linker can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the cell to be targeted. For example, a ligand targeting the liver can be connected to an RNA molecule by a linker including an ester group. Liver cells are rich in esterases, and therefore the linker will be more effectively cut in liver cells than in cell types that are not rich in esterases. Cells of other types that are rich in esterases include cells of the lung, renal cortex, and testis. When targeting cells rich in peptidases (such as liver cells and synovial cells), a linker containing a peptide bond can be used.

[0087] Generally, the suitability of a candidate cleavable joint can be evaluated by testing the ability of a degradable agent (or condition) to cut a candidate joint. It is also desirable to test the ability of a candidate cleavable joint to resist cutting in blood or when in contact with other non-target tissues. Therefore, the relative susceptibility to cutting between the first condition and the second condition can be determined, wherein the first condition is selected to indicate cutting in target cells, and the second condition is selected to indicate cutting in other tissues or biological fluids (such as blood or serum). Evaluation can be carried out in a cell-free system, in cells, in cell cultures, in organs or tissue cultures, or in whole animals. Preliminary evaluation is carried out under cell-free or culture conditions and further evaluation is performed to confirm that the whole animal may be useful. In certain embodiments, compared with in blood or serum (or under in vitro conditions selected to simulate extracellular conditions), the cutting of a useful candidate joint in a cell (or under in vitro conditions selected to simulate intracellular conditions) is at least 2, 4, 10, 20, 50, 70 or 100 times faster.

[0088] In other embodiments, a redox cleavable joint is used. A redox cleavable joint is cleaved when reduced or oxidized. An example of a reductive cleavable group is a disulfide linking group (-SS-). In order to determine whether a candidate cleavable joint is a suitable "reductive cleavable joint" or, for example, whether it is suitable for use with a specific RNAi construct and a specific ligand, one or more methods described herein can be used. For example, a candidate joint can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents known in the art, which simulate the cleavage rate observed in a cell (e.g., a target cell). Candidate joints can also be evaluated under conditions selected to simulate blood or serum conditions. In a particular embodiment, a candidate joint is cut up to 10% in blood. In other embodiments, compared to in blood (or under in vitro conditions selected to simulate extracellular conditions), useful candidate joints are degraded at least 2, 4, 10, 20, 50, 70, or 100 times faster in cells (or under in vitro conditions selected to simulate intracellular conditions).

[0089] In yet other embodiments, the phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that hydrolyze the phosphate group in the cell are enzymes, such as phosphatases in the cell. Examples of phosphate-based cleavable groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-, wherein Rk can be hydrogen or C1-C1 10 Alkyl. Specific examples include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. Another specific example is -OP(O)(OH)-O-. These candidate linkers can be evaluated using methods similar to those described above.

[0090] In other embodiments, the linker may include an acid cleavable group, which is a group that is cleaved under acidic conditions. In some embodiments, the acid cleavable group is cleaved in an acidic environment of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or is cleaved by a reagent such as an enzyme that can act as a general acid. In cells, specific low pH organelles (such as endosomes and lysosomes) can provide a cleavage environment for the acid cleavable group. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid cleavable group may have the general formula -C=NN-, C(O)O, or -OC(O). When the carbon attached to the oxygen (alkoxy group) of the ester is an aryl group, a specific embodiment is a substituted alkyl group or a tertiary alkyl group (such as dimethyl, pentyl, or tert-butyl). These candidates can be evaluated using methods similar to those described above.

[0091] In other embodiments, the linker may comprise an ester-based cleavable group that is cleaved by enzymes in the cell, such as esterases and amidases. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester cleavable group has the general formula -C(O)O- or -OC(O)-. These candidate linkers can be evaluated using methods similar to those described above.

[0092] In other embodiments, the linker may comprise peptide-based cleavable groups that are cleaved by enzymes in the cell, such as peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene groups. Peptide bonds are special types of amide bonds formed between amino acids to produce peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that produce 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)-, where 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.

[0093] Other types of linkers suitable for attaching a ligand to the sense or antisense strand in the RNAi constructs described herein are known in the art and can include, for example, linkers described in: U.S. Patents 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551.

[0094] In certain embodiments, the ligand covalently attached to the sense strand or antisense strand of the RNAi construct comprises a GalNAc moiety, such as a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3' end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5' end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

[0095] In some embodiments, the RNAi construct disclosed herein can be delivered to the target cell or tissue by administering a vector encoding and controlling the intracellular expression of the RNAi construct. "Vector" (also referred to herein as "expression vector") is a composition of material that can be used to deliver the target nucleic acid to the interior of the cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids and viruses. Therefore, 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 can be replicated in living cells, or it can be made synthetically.

[0096] Typically, the vector for expressing the RNAi construct will contain one or more promoters operably linked to the sequence encoding the RNAi construct. The phrases "operably linked", "operably connected" or "under transcriptional control" are used interchangeably herein to indicate the following situation: the promoter is in the correct position and orientation relative to the polynucleotide sequence to control the initiation of transcription of RNA polymerase and the expression of the polynucleotide sequence. "Promoter" refers to a sequence recognized by the cellular synthesis mechanism required for the specific transcription of the gene sequence or the introduced synthesis mechanism. Suitable promoters include, but are not limited to, RNA polI, pol II, HI or U6 RNA pol III, and viral promoters (e.g., human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, and Rous sarcoma virus long terminal repeat). In some embodiments, HI or U6 RNA pol III promoters are used. The promoter may be a tissue-specific or inducible promoter. Of particular interest are liver-specific promoters, such as promoter sequences from human α-1 antitrypsin gene, albumin gene, hemopexin gene, and hepatic lipase gene. Inducible promoters include, for example, promoters regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-PD1-thiogalactoside (IPTG).

[0097] When the RNAi construct includes siRNA, two separate chains (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 connected to the promoter on the first vector, and the sequence encoding the antisense strand is operably connected to the promoter on the second vector. In such an embodiment, the first vector and the second vector are introduced into the target cell together, for example, by infection or transfection, so that the sense strand and the antisense strand, once transcribed, will hybridize in the cell to form siRNA molecules. In another embodiment, the sense strand and the antisense strand are transcribed by two separate promoters located in a single vector. In such an embodiment, the sequence encoding the sense strand can be operably connected to the first promoter, and the sequence encoding the antisense strand can be operably connected to the second promoter, wherein the first promoter and the second promoter are located in a single vector. In one embodiment, the vector comprises a first promoter operably linked to a sequence encoding a siRNA molecule and a second promoter operably linked to the same sequence in the opposite orientation, such that transcription of the sequence from the first promoter results in synthesis of the sense strand of the siRNA molecule and transcription of the sequence from the second promoter results in synthesis of the antisense strand of the siRNA molecule.

[0098] When the RNAi construct comprises shRNA, a sequence encoding a single at least partially self-complementary RNA molecule is operably linked to a promoter to produce a single transcript. In some embodiments, the sequence encoding the shRNA comprises an inverted repeat sequence connected by a linker polynucleotide sequence to produce a stem and loop structure of the shRNA after transcription.

[0099] In some embodiments, the vector encoding the RNAi construct is a viral vector. Various viral vector systems suitable 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; polyoma virus vectors; papilloma virus 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).

[0100] Various vectors suitable for use in the present disclosure, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into vectors, and methods for delivering vectors to target cells are known in the art (see, e.g., 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 DA et al., Nat. Genet., Vol. 33: 401-406, 2003; Brummelkamp et al., Science, Vol. 296: 550-553, 2002; Brummelkamp et al., Cancer Genet., Vol. 34: 551-553, 2003; Brummelkamp et al., Cancer Genet., Vol. 34: 551-553, 2002). 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).

[0101] Composition

[0102] 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 can be used to reduce the expression of SCAP in a subject in need thereof. When clinical applications are contemplated, pharmaceutical compositions and formulations will be prepared in a form suitable for the intended application. Typically, this will require the preparation of compositions that are substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0103] The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other unfavorable reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers, excipients or diluents" include acceptable solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., for the formulation of drugs (such as drugs suitable for administration to humans). The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the RNAi constructs disclosed herein, their use in therapeutic compositions is considered. Supplementary active ingredients may also be incorporated into the composition, provided that they do not inactivate the carrier or RNAi construct of the composition.

[0104] The composition and method for preparing the pharmaceutical composition depend on several standards, including but not limited to route of administration, the type and degree of the disease to be treated or disorder and the dosage to be administered. In certain embodiments, the pharmaceutical composition is prepared based on the expected delivery route. For example, in certain embodiments, the pharmaceutical composition is prepared for parenteral delivery. Parenteral delivery forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal and intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is prepared for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is prepared for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (e.g., a ligand containing GalNAc as described herein).

[0105] 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 a beneficial or desired clinical outcome. In some embodiments, an effective amount is an amount sufficient to reduce the expression of SCAP in a subject's hepatocytes. In some embodiments, an effective amount may be an amount sufficient to only partially reduce SCAP expression (e.g., to a level comparable to that of a wild-type SCAP allele in a human heterozygote).

[0106] The effective amount of the RNAi construct disclosed herein can be from about 0.01mg / kg body weight to about 100mg / kg body weight, about 0.05mg / kg body weight to about 75mg / kg body weight, about 0.1mg / kg body weight to about 50mg / kg body weight, about 1mg / kg to about 30mg / kg body weight, about 2.5mg / kg body weight to about 20mg / kg body weight or about 5mg / kg body weight to about 15mg / kg body weight. The pharmaceutical composition comprising the RNAi construct of an effective amount can be applied weekly, biweekly, monthly, quarterly or semi-annually. The accurate determination of the effective amount considered and the frequency of application can be based on several factors, including patient size, age, sex, obstacle type to be treated (such as steatosis, NAFLD, NASH or cirrhosis), the specific RNAi construct used and route of administration. Conventional methods and / or testing in appropriate animal models can be used to confirm the effective dose of any specific RNAi construct disclosed herein and the estimated value of the half-life in vivo.

[0107] Colloidal dispersion systems (such as macromolecular complexes, nanocapsules, microspheres, beads) and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles and liposomes) can be used as delivery vehicles for the RNAi constructs disclosed herein or vectors encoding such constructs. Commercially available fat emulsions suitable for delivery of nucleic acids include II. III, NUTRILIPID and other similar lipid emulsions. The preferred colloidal system used as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The RNAi construct can be encapsulated in a liposome (such as a cationic liposome). Alternatively, the RNAi construct disclosed herein can be compounded with a lipid (such as a cationic lipid). 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 a colloidal dispersion system are well known in the art. Exemplary formulations are also disclosed in, for example, U.S. Patents 5,783,565; 5,837,533; 5,981,505; 6,127,170; 6,217,900; 6,379,965; 6,383,512; 6,747,014; 7,202,227; and WO 03 / 093449.

[0108] In certain embodiments, the RNAi construct disclosed herein is completely encapsulated in a lipid formulation, for example, to form 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 comprising plasmid DNA encapsulated in lipid vesicles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids and lipids (for example, PEG-lipid conjugates) that prevent particle aggregation. SNALP and SPLP are particularly useful for systemic application because they show extended circulation life after intravenous injection, and accumulate at distal sites (for example, physically separated sites from the site of application). SPLP includes "pSPLP", which includes the encapsulated condensing agent-nucleic acid complexes described in PCT Publication No. WO 00 / 03683. 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 acids present in the nucleic acid-lipid particles are ideally resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed in, for example, U.S. Patents 5,976,567; 5,981,501; 6,534,484; 6,586,410; and 6,815,432; and PCT Publication No. WO 96 / 40964.

[0109] Pharmaceutical compositions suitable for injection include, for example, sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Typically, these preparations are sterile and flow to a degree that is easy to inject. The preparation should be stable under manufacturing and storage conditions, and should prevent the contamination of microorganisms (such as bacteria and fungi). Suitable solvents or dispersion media may 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 a coating (such as lecithin), by maintaining the required particle size (in the case of dispersions) and / or by using a surfactant. The prevention of microbial action can be achieved by various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, isotonic agents (such as, sugar or sodium chloride) may be included in the composition. The extended absorption of injectable compositions can be achieved by including agents that delay absorption (such as, aluminum monostearate and gelatin).

[0110] A sterile injectable solution can be prepared by incorporating a suitable amount of the RNAi construct (alone or complexed with a ligand) together with any other desired ingredients (as described above) into a solvent and then filter sterilizing. Typically, a dispersion is prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains 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 and freeze-drying techniques which yield a powder of one or more active ingredients plus any additional desired ingredients from their previously sterile-filtered solutions.

[0111] The compositions provided herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric or phosphoric acid) or derived from organic acids (e.g., acetic, oxalic, tartaric, mandelic, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide) or derived from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0112] For parenteral administration in an aqueous solution, for example, the solution is typically buffered appropriately and first made isotonic with a diluent such as sufficient saline or glucose. Such aqueous solutions can be used for, for example, intravenous, intramuscular, subcutaneous and intraperitoneal administration. As is known to those of skill in the art, ideally a sterile aqueous medium is used. By way of illustration, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of a subcutaneous injection fluid or injected at the proposed infusion site (see, for example, "Remington’s Pharmaceutical Sciences", 15th Edition, pages 1035 - 1038 and pages 1570 - 1580). For human administration, the preparations should meet the sterility, pyrogenicity, general safety and purity standards required by FDA standards. In certain embodiments of the present disclosure, the pharmaceutical composition comprises a sterile saline solution and the RNAi construct described herein, or consists of the same. In other embodiments, the pharmaceutical composition comprises the RNAi construct described herein and sterile water (e.g., water for injection, WFI), or consists of the same. In still other embodiments, the pharmaceutical composition comprises the RNAi construct described herein and phosphate buffered saline (PBS), or consists of the same.

[0113] In some embodiments, the pharmaceutical composition is packaged with or stored in a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, prefilled syringes, autoinjectors, syringe pumps, portable syringes, and injection pens. Devices for aerosolized or powdered formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present disclosure includes administration devices comprising pharmaceutical compositions for treating or preventing one or more disorders described herein.

[0114] Methods for inhibiting SCAP expression

[0115] The present disclosure also provides methods for inhibiting the expression of the SCAP gene in cells. These methods include contacting the cell with an RNAi construct (e.g., a double-stranded RNAi construct) in an amount that effectively inhibits the expression of SCAP in the cell. Contacting the cell with the RNAi construct (e.g., a double-stranded RNAi construct) can be performed in vitro or in vivo. Contacting the cell with the RNAi construct in vivo includes contacting a cell or a cell population in a subject (e.g., a human subject) with the RNAi construct. Combinations of in vitro and in vivo methods of contacting cells are also within the scope of the present disclosure.

[0116] The present disclosure provides methods for reducing or inhibiting the expression of SCAP in a subject in need thereof, and methods for treating or preventing a condition, disease, or disorder associated with SCAP expression or activity. A "condition, disease, or disorder associated with SCAP expression" refers to a condition, disease, or disorder in which the level of SCAP expression is altered or in which an increase in the level of SCAP expression is associated with an increased risk of developing the condition, disease, or disorder.

[0117] As discussed above, contacting cells can be direct or indirect. In addition, contacting cells can be achieved via a targeting ligand, including any ligand described herein or known in the art. In a preferred embodiment, the targeting ligand is a carbohydrate moiety (e.g., a triantennary GalNAc ligand (GalNAc3)) or any other ligand that guides the RNAi construct to the target site.

[0118] In one embodiment, contacting a cell with an RNAi comprises "introducing" or "delivering the RNAi into a cell" by facilitating or enabling uptake or absorption into the cell. The uptake or uptake of the RNAi can occur through an unassisted diffusion process or an active cellular process or with the aid of an adjuvant or device. For example, for in vivo introduction, the RNAi can be injected into a tissue site or administered systemically. In vitro introduction into a cell can be achieved using methods known in the art, such as electroporation and lipofection. Additional methods are described below and / or are known in the art.

[0119] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "repress," and other similar terms, and includes any level of inhibition.

[0120] The phrase "inhibiting the expression of SCAP" is intended to refer to the inhibition of the expression of any SCAP gene (e.g., a mouse SCAP gene, a rat SCAP gene, a monkey SCAP gene, or a human SCAP gene) as well as variants or mutants of the SCAP gene. Thus, in the context of a genetically manipulated cell, a cell population, or an organism, the SCAP gene may be a wild-type SCAP gene, a mutant SCAP gene, or a transgenic SCAP gene.

[0121] "Inhibiting the expression of the SCAP gene" includes any level of inhibition of the SCAP gene, for example, at least partial suppression of the expression of the SCAP gene. The expression of the SCAP gene can be assessed based on the level or change in level of any variable associated with the expression of the SCAP gene (e.g., SCAP mRNA level or SCAP protein level). The level can be assessed in a single cell or a cell population (including, for example, a sample derived from a subject).

[0122] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with SCAP expression compared to a control level. The control level can be any type of control level used in the art, for example, a baseline level before administration, or a level determined by a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control). In some embodiments, 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%.

[0123] Inhibition of SCAP gene expression can be demonstrated by a decrease in the amount of mRNA expressed by a first cell or cell population (such cells can be present, for example, in a sample derived from a subject) in which SCAP is transcribed and which has been treated (e.g., by contacting one or more cells with an RNAi construct disclosed herein, or by administering an RNAi construct disclosed herein to a subject in which such cells are or have been present) such that expression of the SCAP gene is inhibited compared to a second cell or cell population (one or more control cells) that is substantially identical to the first cell or cell population but has not been so treated. Inhibition can be assessed by expressing the mRNA level in the treated cells as a percentage of the mRNA level in the control cells using the following formula:

[0124]

[0125] Alternatively, inhibition of SCAP gene expression can be assessed based on a reduction in a parameter functionally related to SCAP gene expression (e.g., SCAP protein expression or SREBP family protein activity). SCAP gene silencing can be determined in any cell that expresses SCAP endogenously or recombinantly by any assay known in the art.

[0126] Inhibition of SCAP protein expression can be represented by a decrease 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 explained above, for the purpose of assessing mRNA repression, inhibition of protein expression levels in treated cells or cell populations can be similarly expressed as a percentage of protein levels in control cells or cell populations.

[0127] Control cells or cell populations that can be used to assess inhibition of SCAP gene expression include cells or cell populations that have not been contacted with the RNAi constructs disclosed herein. For example, control cells or cell populations can be derived from a single subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi construct.

[0128] The level of SCAP mRNA expressed by a cell or cell population or the level of circulating SCAP mRNA can be determined using any method known in the art for assessing mRNA expression, such as those mentioned above. In some embodiments, the expression level of SCAP in a sample is determined by detecting a transcribed polynucleotide or portion thereof (e.g., mRNA of a SCAP gene). In this regard, RNA can be extracted from cells using, for example, 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 RNA hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. [Nucleic Acids Research], 12:7035), Northern blots, in situ hybridization, and microarray analysis. Circulating SCAP mRNA can be detected using the methods described in WO 2012 / 177906.

[0129] In one embodiment, nucleic acid probes are used to determine the expression level of SCAP. 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 of ordinary skill in the art, or derived from an appropriate biological agent. The probe can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0130] The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern blot analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method of determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with SCAP mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane (such as nitrocellulose). In an alternative embodiment, one or more probes are immobilized on a solid surface and the mRNA is contacted with the one or more probes, for example, in an Affymetrix gene chip array. Those skilled in the art can readily adapt known mRNA detection methods for determining SCAP mRNA levels.

[0131] Alternative methods for determining the level of SCAP expression in a sample involve, for example, nucleic acid amplification of mRNA in the sample and / or the use of a reverse transcriptase (to prepare cDNA), such as by RT-PCR (see, e.g., U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustaining sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA [National Academy of Sciences of the United States of America] 91:189-193), or cDNA sequencing (Cell Signaling Technology, Inc., New York, NY). [Proc. Natl. Acad. Sci. USA] 87:1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA [Proc. Natl. Acad. Sci. USA] 86:1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology [Biotechnology] 6:1197), rolling circle replication (Lizardi et al., supra; and U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules if such molecules are present in very low numbers. In some aspects of the present disclosure, quantitative fluorescent RT-PCR (i.e., TAQMAN TM The expression level of SCAP can be determined by a method of using a nucleic acid probe in a solution. In some embodiments, the expression level of SCAP is determined by using a membrane blot (e.g., for hybridization analysis, such as Northern blot, Southern blot, spot, etc.) or a microwell, sample tube, gel, bead or fiber (or any solid support containing bound nucleic acid) to monitor the expression level of SCAP mRNA (see, e.g., U.S. Patents 5,445,934; 5,677,195; 5,770,722; 5,744,305; and 5,874,219). The determination of the expression level of SCAP can also include the use of a nucleic acid probe in solution. In certain embodiments, the mRNA expression level is assessed using a branched DNA (bDNA) assay or real-time PCR (qPCR).

[0132] 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), superdiffusion chromatography, fluid or gel precipitin reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0133] In some embodiments, the efficacy of the methods disclosed herein can be monitored by detecting or monitoring a reduction in symptoms of a disease associated with SCAP, such as gastrointestinal pain, dyspnea, hypertension, or swelling of the extremities, face, throat, upper respiratory tract, abdomen, trunk, and genitals. These symptoms can be assessed in vitro or in vivo using any method known in the art.

[0134] In some embodiments, an RNAi construct or a composition comprising an RNAi construct is administered to a subject so that the RNAi construct is delivered to a specific site within the subject. The inhibition of SCAP expression can be assessed using measurements of the level or change in the level of SCAP mRNA or SCAP protein in a sample of fluid or tissue derived 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 may also be a sub-portion or subgroup of cells from any of the above sites. The site may also include cells expressing a specific type of receptor.

[0135] Methods of treating or preventing SCAP-related diseases

[0136] The present disclosure provides therapeutic and preventive methods comprising administering an RNAi construct as described herein, a composition (e.g., a pharmaceutical composition) comprising an RNAi construct, or a vector comprising an RNAi construct to a subject suffering from a disease, disorder, and / or condition associated with SCAP or susceptible to developing a disease, disorder, and / or condition associated with SCAP. Non-limiting examples of diseases associated with SCAP include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocyte necrosis, liver fibrosis, obesity, non-alcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and hyperlipidemia. In one embodiment, the disease associated with SCAP is NAFLD. In another embodiment, the disease associated with SCAP is NASH. In another embodiment, the disease associated with SCAP is fatty liver (steatosis). In another embodiment, the disease associated with SCAP is insulin resistance.

[0137] In certain embodiments, the disclosure provides a method for reducing the expression of SCAP in a patient in need thereof, the method comprising administering to the patient any RNAi construct described herein. As used herein, the term "patient" refers to a mammal, including a human, and is used interchangeably with the term "subject". The level of SCAP expression in the patient's hepatocytes is desirably reduced after administration of the RNAi construct compared to the level of SCAP expression in the patient who did not receive the RNAi construct.

[0138] The methods disclosed herein can be used to treat subjects with diseases associated with SCAP, such as subjects who will benefit from a reduction in SCAP gene expression and / or SCAP protein production. In one aspect, the disclosure provides methods for reducing the level of SCAP gene expression in subjects with non-alcoholic fatty liver disease (NAFLD). In another aspect, the disclosure provides methods for reducing the level of SCAP protein in subjects with NAFLD.

[0139] The treatment methods (and uses) disclosed herein include administering to a subject (eg, a human) a therapeutically effective amount of the disclosed RNAi construct targeting the SCAP gene, a pharmaceutical composition comprising the RNAi construct, or a vector comprising the RNAi construct.

[0140] In one aspect, the disclosure provides a method for preventing at least one symptom in a subject with NAFLD, such as the presence of elevated hedgehog signaling pathways, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider veins, yellowing of the skin and eyes (jaundice), itching, fluid accumulation and swelling of the legs (edema), swelling of the abdomen (ascites), and mental confusion. These methods include administering to the subject a preventive effective amount of an RNAi construct (e.g., siRNA), a pharmaceutical composition comprising an RNAi construct, or a vector encoding an RNAi construct, thereby preventing at least one symptom in a subject with a disorder that will benefit from a reduction in SCAP gene expression. A "preventive effective amount" refers to an amount that effectively achieves a desired preventive outcome (e.g., preventing the onset of a disease) at the necessary dosage and time period.

[0141] In another aspect, the disclosure provides the use of a therapeutically effective amount of an RNAi construct disclosed herein for treating a subject, such as a subject that will benefit from reduction and / or inhibition of SCAP gene expression. In another aspect, the disclosure provides the use of an RNAi construct (e.g., siRNA) targeting a SCAP gene, or a pharmaceutical composition comprising an RNAi construct targeting a SCAP gene in the manufacture of a medicament for treating a subject (e.g., a subject that will benefit from reduction and / or inhibition of SCAP gene expression and / or SCAP protein production, such as a subject with a disorder (e.g., a disease associated with SCAP) that will benefit from reduction of SCAP gene expression).

[0142] The present disclosure provides the use of the RNAi constructs (e.g., siRNA) disclosed herein for preventing at least one symptom in a subject with a disorder that can benefit from 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.

[0143] In additional aspects, the present disclosure provides 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 benefits from reduction and / or inhibition of SCAP gene expression and / or SCAP protein production, such as a SCAP-associated disease.

[0144] In one embodiment, a subject having a disease associated with SCAP, such as non-alcoholic fatty liver disease (NAFLD), is administered an RNAi construct targeting SCAP such that when the RNAi construct is administered to the subject, expression of the SCAP gene, e.g., in the subject's cells, tissues, blood or other tissues or body fluids, 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%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 , 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.

[0145] The methods and uses disclosed herein include administering the compositions described herein such that the reduction in expression of the target SCAP gene lasts for any suitable amount of time, such as 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, the expression of the target SCAP gene is reduced for an extended duration (e.g., at least about 2, 3, 4, 5, 6, 7 days or longer, such as about 1 week, 2 weeks, 3 weeks, or about 4 weeks or longer).

[0146] Administration of RNAi constructs according to the methods and uses disclosed herein can result in a reduction in the severity, signs, symptoms and / or markers of such diseases or disorders in patients with diseases associated with SCAP (e.g., NAFLD). In this context, "reduction" means a statistically significant reduction in such levels. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100%. The efficacy of treating or preventing a disease can be assessed, for example, by measuring the level of any other measurable parameter of a given disease to be treated or targeted for prevention, such as disease progression, disease remission, symptom severity, pain relief, quality of life, drug dosage required to maintain a therapeutic effect, disease markers, or other measurable parameters applicable to a given disease to be treated or targeted for prevention. Monitoring the efficacy of treatment or prevention by measuring any one or any combination of such parameters is within the capabilities of those of ordinary skill in the art. For example, the efficacy of treating NAFLD can be assessed, for example, by regularly monitoring NAFLD symptoms, liver fat levels, or expression of downstream genes. Comparison of subsequent readings with initial readings provides the physician with an indication of whether the treatment is effective. With respect to the administration of RNAi or pharmaceutical compositions thereof targeting SCAP, "effectively targeting" a disease associated with SCAP indicates that administration in a clinically appropriate manner results in a beneficial effect in at least a statistically significant portion of patients, such as improvement of symptoms, cure, alleviation of disease, extension of life, improvement in quality of life, or other effects generally considered positive by physicians familiar with treating NAFLD and / or diseases associated with SCAP and associated causes.

[0147] When one or more parameters of the disease state are statistically significantly improved, or because of the failure to deteriorate or develop symptoms that would otherwise be expected, the therapeutic or preventive effect is evident. As an example, at least 10%, preferably at least 20%, 30%, 40%, 50% or more favorable changes in measurable disease parameters can indicate effective treatment. Experimental animal models for given diseases as known in the art can also be used to judge the efficacy of a given RNAi construct or a formulation of the construct. When using an experimental animal model, when a statistically significant reduction in markers or symptoms is observed, the therapeutic efficacy is demonstrated.

[0148] Any therapeutically effective amount of RNAi construct can be used to the subject. The exemplary range of the therapeutically effective amount of RNAi construct includes but is not limited to from about 0.01mg / kg body weight to about 100mg / kg body weight, about 0.05mg / kg body weight to about 75mg / kg body weight, about 0.1mg / kg body weight to about 50mg / kg body weight, about 1mg / kg to about 30mg / kg body weight or about 2.5mg / kg body weight to about 20mg / kg body weight. This disclosure also encompasses values ​​and scopes in the middle of the narrated range.

[0149] Administration of an RNAi construct or a composition comprising the same can reduce the presence of SCAP protein levels (e.g., in 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%, 99%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0150] Prior to administering a full dose of RNAi, a smaller dose (e.g., 5% infusion) may be administered to the patient and adverse reactions (e.g., allergic reactions) monitored. In another example, the patient may be monitored for unwanted immunostimulatory effects, such as elevated cytokine (e.g., TNF-α or INF-α) levels.

[0151] Due to the inhibitory effect on SCAP expression, the composition according to the present disclosure or the pharmaceutical composition prepared therefrom can enhance the quality of life of patients suffering from diseases associated with SCAP (eg, NAFLD).

[0152] The RNAi disclosed herein can be administered in a "naked" form, where the modified or unmodified RNAi construct is directly suspended in an aqueous or suitable buffer solvent as "free RNAi". Free RNAi is administered in the absence of a pharmaceutical composition. Free RNAi can be in a suitable buffer solution. The buffer solution may contain acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolality of the buffer solution containing RNAi can be adjusted to make it suitable for administration to a subject.

[0153] Alternatively, the RNAi constructs disclosed herein can be administered as a pharmaceutical composition, such as a liposomal formulation.

[0154] Subjects who would benefit from reduction and / or inhibition of SCAP gene expression are those subjects suffering from non-alcoholic fatty liver disease (NAFLD) and / or another SCAP-associated disease or disorder as described herein or otherwise known in the art.

[0155] The present disclosure further provides methods and uses of RNAi constructs or pharmaceutical compositions thereof in combination with other drugs and / or other therapeutic approaches for treating subjects who would benefit from reduction and / or inhibition of SCAP gene expression (e.g., subjects suffering from a disease associated with SCAP), such as known drugs and / or known therapeutic approaches, such as those currently used to treat these disorders.

[0156] For example, in certain embodiments, an RNAi construct targeting a SCAP gene is administered in combination with an agent, for example, useful for treating a disease associated with SCAP. For example, additional therapeutic agents and therapeutic methods suitable for treating a subject who would benefit from a reduction in SCAP expression (e.g., a subject suffering from a disease associated with SCAP) include RNAi constructs targeting different portions of a SCAP gene, therapeutic agents, and / or procedures for treating a disease associated with SCAP, or a combination of any of the foregoing.

[0157] In certain embodiments, a first RNAi construct targeting a portion of a SCAP gene is administered in combination with a second RNAi construct targeting a different portion of the SCAP gene. For example, a first RNAi construct may include a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all nucleotides of the first sense strand and substantially all nucleotides of the first antisense strand are modified nucleotides, wherein the first sense strand is conjugated to a ligand attached at the 3'-end, and wherein the ligand is one or more GalNAc derivatives attached through a divalent or trivalent branched linker; and a second RNAi construct may include a second sense strand and a second antisense strand forming a double-stranded region, wherein substantially all nucleotides of the second sense strand and substantially all nucleotides of the second antisense strand are modified nucleotides, wherein the second sense strand is conjugated to a ligand attached at the 3'-end, and wherein the ligand is one or more GalNAc derivatives attached through a divalent or trivalent branched linker. In one embodiment, all nucleotides of the first and second sense strands and / or all nucleotides of the first and second antisense strands comprise modifications. The modified nucleotide may be any one or combination of the modified nucleotides described herein.

[0158] 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 can be administered in combination with an RNAi construct targeting the patatin-like phospholipase domain 3 (PNPLA3) gene. The I148M mutant PNPLA3 protein is a therapeutic target for the treatment of non-alcoholic fatty liver disease (NASH) with strong human gene validation. The expression of PNPLA3 I148M leads to the accumulation of excess liver fat and drives the increased incidence, progression and severity of phenotypes associated with non-alcoholic fatty liver disease (NAFLD). A first RNAi construct targeting the SCAP gene and a second RNAi construct targeting a different gene (e.g., the PNPLA3 gene) can be administered as part of the same pharmaceutical composition. Alternatively, a first RNAi construct targeting the SCAP gene and a second RNAi construct targeting a different gene (e.g., the PNPLA3 gene) can be administered as part of different pharmaceutical compositions.

[0159] The RNAi construct and the additional therapeutic agent and / or treatment can be administered at the same time and / or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at a separate time and / or by another method known in the art or described herein.

[0160] The present disclosure also provides methods for reducing and / or inhibiting the expression (gene or protein expression) of SCAP in cells using RNAi constructs disclosed herein and / or compositions containing RNAi constructs. In yet other aspects, the use of RNAi constructs disclosed herein and / or compositions containing RNAi constructs for the manufacture of drugs is provided, and the drugs are used to reduce and / or inhibit the expression of SCAP genes in cells. In still other aspects, the present disclosure provides RNAi and / or compositions containing RNAi constructs described herein for reducing and / or inhibiting the production of SCAP proteins in cells. In yet other aspects, the use of RNAi constructs described herein and / or compositions containing RNAi constructs for the manufacture of drugs is provided, and the drugs are used to reduce and / or inhibit the production of SCAP proteins in cells. These methods and uses include contacting cells with RNAi constructs disclosed herein (e.g., siRNA), and maintaining the cells for a sufficient time to obtain degradation of mRNA transcripts of the SCAP gene, thereby inhibiting the expression of the SCAP gene or inhibiting the production of SCAP proteins in cells. The reduction in gene expression can be assessed by any method known in the art or described herein for determining mRNA or protein levels.

[0161] In the methods and uses disclosed herein, cells can be contacted in vitro or in vivo, i.e., cells can be outside a subject (e.g., in cell culture) or in a subject. Cells suitable for treatment using the methods disclosed herein can be any cells expressing the SCAP gene, such as cells from a subject suffering from NAFLD or cells comprising an expression vector comprising the SCAP gene or a portion of the SCAP gene. Suitable cells for use in the disclosed methods include, for example, mammalian cells, such as primate cells (e.g., human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (e.g., cattle cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamsters, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell.

[0162] The expression of the SCAP gene can be inhibited in the cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, %, 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%.

[0163] SCAP protein production can be inhibited in a cell by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, %, 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%.

[0164] The in vivo methods and uses disclosed herein may include administering to a subject a composition containing an RNAi construct, wherein the RNAi construct includes a nucleotide sequence complementary to at least a portion of an RNA transcript of a 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, including but not limited to subcutaneous, intravenous, oral, intraperitoneal or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal and topical (including oral and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection. In one embodiment, the composition is administered by subcutaneous injection.

[0165] In some embodiments, administration is via a long-acting injection. Long-acting injections can release RNAi constructs in a consistent manner over an extended period of time. Therefore, long-acting injections can reduce the frequency of administration required to obtain a desired effect (e.g., a desired SCAP inhibition, or a therapeutic or preventive effect). Long-acting injections can also provide more consistent serum concentrations. Long-acting injections can include subcutaneous injections or intramuscular injections. In some embodiments, long-acting injections are subcutaneous injections.

[0166] In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. Infusion pumps can be used for intravenous, subcutaneous, arterial, or epidural infusions. 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.

[0167] The mode of administration can be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route of administration and site of administration can be selected to enhance targeting.

[0168] These methods and uses include administering to a mammal (e.g., a human) a composition comprising an RNAi construct (e.g., siRNA) targeting a SCAP gene in a mammalian cell, and maintaining the mammal for a sufficient time to obtain degradation of the mRNA transcript of the SCAP gene, thereby inhibiting the expression of the SCAP gene in the mammal. The reduction of gene expression and / or protein expression can be assessed in a sample obtained from a subject to which the RNAi construct has been administered by any method known in the art or described herein. In one embodiment, a tissue sample serves as a tissue material for monitoring the reduction of SCAP gene and / or protein expression. In another embodiment, a blood sample serves as a tissue material for monitoring the reduction of SCAP gene and / or protein expression.

[0169] In some embodiments, verification of RISC-mediated cleavage of a target mRNA (e.g., SCAP mRNA) in vivo following administration of an RNAi construct can be assessed by performing 5'-RACE or modifications of protocols known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38(3) p-el9; and Zimmermann et al. (2006) Nature 441: 111-4).

[0170] It should be understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by replacing uracil bases in the sequence with thymine bases. Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by replacing thymine bases in the sequence with uracil bases. This disclosure encompasses deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences of mixtures of deoxyribonucleotides and ribonucleotides containing all sequences disclosed herein.

[0171] Additionally, any nucleic acid sequence disclosed herein can be modified with any combination of chemical modifications. One of ordinary skill in the art will readily appreciate that in some cases, the nomenclature describing a modified polynucleotide, such as "RNA" or "DNA," is arbitrary. For example, a polynucleotide comprising a nucleotide and a thymine base having a 2'-OH substituent on the ribose sugar can be described as a DNA molecule having a modified sugar (2'-OH for the natural 2'-H of DNA) or as an RNA molecule having a modified base (thymine (methylated uracil) for the natural uracil of RNA).

[0172] Therefore, the nucleic acid sequences provided herein (including but not limited to those set forth in the sequence table) are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to such nucleic acids with modified nucleoside bases. As another example and being non-limiting, the polynucleotides with the sequence "ATCGATCG" encompass any polynucleotides with such sequences that are modified or unmodified, including but not limited to such compounds comprising RNA bases, such as those compounds with the sequence "AUCGAUCG" and those compounds with some DNA bases and some RNA bases (such as "AUCGATCG"), and polynucleotides with other modified bases (such as "ATmeCGAUCG"), wherein meC indicates a cytosine base comprising a methyl group at the 5-position.

[0173] The following examples, including experiments performed and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

[0174] Examples

[0175] All animal experiments described in this article were approved by the Institutional Animal Care and Use Committee (IACUC) of Amgen and were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National Research Council (US)). Committee for the Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (US), and National Academies Press (US) (2011) Guide for the care and use of laboratory animals. 8th edition, National Academies Press, Washington, DC. Mice were housed individually in an air-conditioned room at 22°C ± 2°C with a 12-hour light and 12-hour dark cycle (0600-1800 hours). Unless otherwise indicated, animals were given free access to regular food (Envigo, 2920X; or food as otherwise specified) and water (purified by reverse osmosis) via an automatic watering system. At termination, blood was collected by cardiac puncture under deep anesthesia, and then euthanasia was performed by secondary physical methods according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

[0176] Example 1: Selection, design and synthesis of modified SCAP siRNA molecules

[0177] Bioinformatics analysis of human SREBP cleavage activation protein (SCAP) transcripts was used to identify and select the optimal sequence of therapeutic siRNA molecules targeting the SCAP gene. The siRNA trigger was prepared as a chemically modified siRNA duplex consisting of a sense (follower) strand and an antisense (guide) strand. Both strands were 18-23 nucleotides in length with a 3' overhang of two base pairs. The siRNA was modified so that the natural 2'-OH in the ribose of each nucleotide was replaced by a 2'-OMe or 2'-F group, and the two phosphodiesterase internucleotide bonds at each end of the two strands were replaced by thiophosphates to reduce degradation by exonucleases. The resulting unmodified and modified antisense and sense siRNA sequences are shown in Table 1.

[0178] Nucleotide sequences in Table 1 and elsewhere in this application are listed according to the following symbols: 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 = diol nucleic acid; sGNA = diol nucleic acid with 3' phosphorothioate; LNA = locked nucleic acid. Insertion of an "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothioate diester group (eg, a phosphorothioate internucleotide bond). Unless otherwise indicated, all other nucleotides are linked by a 3'-5' phosphodiester group.

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[0190] Example 2: Efficacy of selected SCAP siRNA molecules in RNA FISH assay

[0191] The siRNA molecules synthesized in Example 1 were screened in a fluorescence in situ hybridization assay targeting RNA molecules (RNA FISH) to determine the IC 50 and maximum activity value.

[0192] RNA FISH assay was performed to measure SCAP mRNA knockdown by test siRNA. Hep3B cells (purchased from ATCC) were cultured in minimum essential medium (MEM, Corning) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Corning). siRNA transfection was performed as follows: 1 μL of test siRNA and 4 μL of plain MEM were added to PDL-coated CellCarrier-384Ultra assay plates (PerkinElmer) by BioMek FX (Beckman Coulter). Then, 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) pre-diluted in plain MEM (0.035 μL RNAiMAX in 5 μL MEM) was dispensed into the assay plate by a Multidrop Combi reagent dispenser (Thermo Fisher Scientific). After incubating the siRNA / RNAiMAX mixture at room temperature (RT) for 20 minutes, 30 μL of Hep3B cells (2000 cells / well) in MEM supplemented with 10% FBS and 1% PS were added to the transfection complex using a Multidrop Combi reagent dispenser and the assay plates were kept at RT for 20 minutes before being moved to an incubator. The cells were incubated at 37°C and 5% CO2 for 72 hours.

[0193] The PCR products were analyzed using Affymetrix according to the manufacturer's protocol. RNA FISH assays were performed using the View RNA HC Screening Assay Kit (QVP0011), Affymetrix View HC Signal Amplification Kit 3-plex (QVP0213), and Affymetrix gene-specific probes (SCAP Human 0.33 mL View RNA Type 6 (650 markers) VA6-20279-01 and PPIB Human 0.44 mL View RNA Type 1 (488 markers) VA1-10148-01).

[0194] First, the plate is rehydrated with continuous 100%, 70% and 50% ethanol washing. The cells are then washed with PBS and then permeabilized and digested with proteases according to the kit instructions. The target working probe set is prepared according to the manufacturer's protocol, added to the wells, and incubated for 3 hours at 40°C. Continuous hybridization using the working probe set, working preamplifier, working amplifier and working LP is carried out according to the manufacturer's protocol. Finally, nuclear counterstaining (Hoechst 33342 and CellMask Blue; Molecular Probes) is applied. The plate is incubated at room temperature for 30 minutes, washed with PBS, covered with 80 μl of PBS, and then the plate is sealed for imaging.

[0195] 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 type 1 probes, and the 647 channel for type 6 probes.

[0196] RNA FISH data were analyzed using Columbus software and images were generated using Genedata Screener. Images were analyzed to obtain average spot counts per cell. Spot counts were normalized using high (containing phosphate buffered saline, Corning) and low (no target probe pair) control wells. Normalized values ​​relative to total siRNA concentration were plotted and data were fitted to a four-parameter sigmoid model in Genedata Screener (Genedata) to obtain IC 50 and maximum activity.

[0197] The results of the assay are shown in Table 2. SCAP activity is expressed as a percentage of knockdown compared to the control. Negative values ​​indicate a decrease in SCAP levels. Several siRNA triggers were additionally tested, and the results are shown in the last column of Table 2.

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[0206] Example 3: Efficacy screening of selected SCAP siRNA molecules in a mouse model

[0207] Male Balbc mice aged 7-8 weeks and greater than 20 grams were obtained from Charles River Laboratories (Charles Rivers Laboratory, Inc, MA). For robust hepatic expression, mice were dosed with AAV8 viral vectors. 1 x 10 12 Each animal is administered with individual virus particles. AAV vectors encode reporter constructs based on luciferase genes. After 10 minutes of intraperitoneal administration of Xenolight Rediject D-luciferin substrate (Perkin Elmer), luciferase activity is analyzed by measuring the light signal generated from luciferin / luciferase reaction. These light signals are indicated as flux. Bioluminescence imaging (BLI) is performed using the IVIS Lumina S5 Spectrum preclinical in vivo imager from Perkin Elmer. BLI image analysis is performed by placing a small and identical region of interest (ROI) on the liver, and Living Image software 4.7.2 is used for analysis.

[0208] To monitor the activity of SCAP siRNA, the 3'UTR region of the luciferase reporter gene includes a specific SCAP mRNA sequence, to which the siRNA can bind and initiate degradation of the luciferase reporter gene. Two weeks after AAV administration, mice were imaged for baseline luciferase activity. Animals were then randomized according to baseline image intensity and grouped into 5 animals per siRNA. The next day, mice were subcutaneously administered with 3 mg / kg body weight (mpk) of SCAP siRNA conjugated with triantennary GalNAc (GalNAc3) at the 5' end of the sense strand. Four weeks after siRNA administration, BLI imaging was performed to monitor luciferase activity. The siRNA efficacy at week 4 is shown in Table 3 as % silencing data, which is calculated by normalizing the ratio of the 4th week (Wk4) flux of each siRNA group to the PBS group: baseline flux. A total of 158 triggers were tested, several of which included different chemical modifications.

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[0216] All publications, patents, and patent applications mentioned in this specification are incorporated herein 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, citation of references herein should not be construed as an admission that such references are prior art to the present invention. In the event that any definitions or terms provided in references incorporated by reference differ from the terms and discussions provided herein, the present terms and definitions shall prevail.

[0217] The foregoing written description is considered sufficient to enable one of ordinary skill in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode considered by the inventors. However, it will be understood that no matter how detailed the foregoing may appear in text, the invention may be implemented in many ways, and the invention should be interpreted in accordance with the appended claims and any equivalents thereof.

Claims

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of at least 15 consecutive nucleotides having no more than 3 nucleotide differences from the antisense sequences listed in Table 1, and wherein the RNAi construct inhibits the expression of SREBP cleavage activating protein (SCAP) mRNA.

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

3. The RNAi construct of claim 1 or claim 2, wherein the duplex region is about 17 to about 24 base pairs in length.

4. The RNAi construct of any one of claims 1-3, wherein the duplex region is about 19 to about 21 base pairs in length.

5. The RNAi construct of claim 4, wherein the duplex region is 19 base pairs in length.

6. The RNAi construct of any one of claims 1-5, wherein the sense strand and the antisense strand are each about 15 to about 30 nucleotides in length.

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

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

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

10. The RNAi construct of any one of claims 1-9, comprising at least one blunt end.

11. The RNAi construct of any one of claims 1-10, comprising at least one nucleotide overhang having 1 to 4 unpaired nucleotides.

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

13. The RNAi construct of claim 11 or 12, wherein the RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' end of both the sense strand and the antisense strand.

14. The RNAi construct of any one of claims 11-13, wherein the nucleotide overhang comprises a 5'-UU-3' dinucleotide or a 5'-dTdT-3' dinucleotide.

15. The RNAi construct of any one of claims 1-14, wherein the RNAi construct comprises at least one modified nucleotide.

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

17. The RNAi construct of claim 15, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a diol nucleic acid, an inverted nucleotide, or a combination thereof.

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

19. The RNAi construct of any one of claims 15-18, wherein all nucleotides in the sense strand and the antisense strand are modified nucleotides.

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

21. The RNAi construct of any one of claims 1-20, comprising at least one phosphorothioate internucleotide bond.

22. The RNAi construct of claim 21, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide bonds at the 3' end of the antisense strand.

23. The RNAi construct of claim 21, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5' end of the sense strand.

24. The RNAi construct of any one of claims 1-23, wherein the antisense strand comprises a sequence selected from the antisense sequences listed in Table 1.

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

26. The RNAi construct of any one of claims 1-25, wherein the RNAi construct is any one of the duplex compounds listed in Table 1.

27. The RNAi construct of any one of claims 1-26, which is a small interfering RNA (siRNA).

28. The RNAi construct of any one of claims 1-27, wherein the RNAi construct reduces the level of SCAP expression in liver cells following incubation with the RNAi construct compared to the level of SCAP expression in liver cells that have been incubated with a control RNAi construct.

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

30. The RNAi construct of any one of claims 1-29, wherein the RNAi construct inhibits SCAP expression by at least 40% at 5 nM in Hep3B cells in vitro.

31. The RNAi construct of any one of claims 1-29, wherein the RNAi construct inhibits SCAP expression by at least 40% at 5 nM in HepG2 cells in vitro.

32. The RNAi construct of any one of claims 1-31, wherein the RNAi construct inhibits the expression of SCAP in Hep3B cells with an IC50 of less than about 5 nM.

33. The RNAi construct of any one of claims 1-31, wherein the RNAi construct inhibits expression of SCAP in HepG2 cells with an IC50 of less than about 5 nM.

34. The RNAi construct of any one of claims 1-33, further comprising a ligand that binds to one or more proteins expressed on the surface of liver cells.

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

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

37. A method for reducing the expression of SCAP in a patient in need thereof, the method comprising administering to the patient the composition of claim 35.

38. The method of claim 36 or claim 37, wherein the expression level of SCAP in hepatocytes is reduced in the patient following administration of the RNAi construct compared to the expression level of SCAP in a patient who did not receive the RNAi construct.

39. The method of any one of claims 36-38, wherein the patient has non-alcoholic fatty liver disease (NAFLD).

40. The method of claim 39, wherein the patient has nonalcoholic steatohepatitis (NASH).

41. The RNAi construct of any one of claims 1-34 or the composition of claim 35 for use in treating NAFLD.

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

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