Rnai constructs for inhibiting SCAP expression and methods of using same

Abstract

The present invention relates to RNAi constructs for reducing the expression of the SCAP gene. Also described are methods of using such RNAi constructs to treat or prevent liver disease, non-alcoholic fatty liver disease (NAFLD).

Description

[0001] This application is a divisional application of an invention patent with an application date of June 1, 2020, application number 202080041291.2, and invention name “RNAI construct for inhibiting SCAP expression and method of use thereof”. Technical Field

[0002] The present invention relates to compositions and methods for regulating the hepatic expression of sterol regulatory element binding protein (SREBP) cleavage activation protein (SCAP). In particular, the present invention relates to nucleic acid-based therapeutic agents that reduce SCAP expression by RNA interference (RNAi), and methods of using such nucleic acid-based therapeutic agents to treat or prevent liver diseases such as non-alcoholic fatty liver disease (NAFLD).

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 854,433, filed May 30, 2019, which is incorporated herein by reference in its entirety. Background Art

[0005] Nonalcoholic fatty liver disease (NAFLD), which encompasses a spectrum of liver pathologies, is the most common chronic liver disease in the world. Its prevalence has doubled in the past 20 years and is currently estimated to affect approximately 20% of the world's population (Sattar et al. (2014) BMJ 349:g4596; Loomba and Sanyal (2013) Nature Reviews Gastroenterology & Hepatology 10(11):686-690; Kim and Kim (2017) Clin Gastroenterol Hepatol 15(4):474-485; Petta et al. (2016) Dig Liver Dis 48(3):333-342). NAFLD begins with the accumulation of triglycerides in the liver, which is defined as the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes in individuals who 1) do not have a history of heavy 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, known as steatosis, causes inflammation and hepatocellular damage, leading to a more advanced stage of the disease known as nonalcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, an estimated 75 to 100 million Americans have NAFLD; NASH accounts for approximately 10% to 30% of NAFLD diagnoses (Rinella, supra; Younossi et al. (2016) Hepatology 64(5):1577-1586).

[0006] SCAP (SREBP cleavage activation protein) is the only known post-transcriptional regulator of the SREBP family of transcription factors. The SREBP (sterol response element binding protein) family plays an important role in the regulation of de novo lipogenesis and TG accumulation in the liver. In the ER, SREBP is synthesized in the form of an inactive precursor. Immediately after synthesis, SCAP forms a complex with SREBP and escorts the transport of SREBP to the Golgi vesicles. SREBP is then 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 (Brown, MS and Goldstein, JL (1997) Cell [Cell] 89, 331-340). Targeted silencing of SCAP has been proposed to prevent processing and downstream transcriptional changes of active SREBP.

[0007] The SREBP family of proteins includes three isoforms: SREBP-1a, SREBP-1c, and SREBP-2, which have different but overlapping functions. SREPB-1c is abundant in the liver and primarily activates fatty acid and TG synthesis. Germline deletion of SREBP-1 exhibits a concomitant increase in SREBP-2 levels, which compensates for the loss of SREBP-1.

[0008] SREBP-2 drives cholesterol production and low-density lipoprotein (LDL) processing by activating the LDL receptor (LDLR). SREBP-2 also regulates PCSK9, a secreted protein that interacts with the LDLR to promote its degradation and reduce cholesterol uptake. Loss of SCAP / SREBP maintains protein levels of the LDLR. SREBP1c is also the only known transcriptional regulator of PNPLA3. The PNPLA3 polymorphism rs738409 (I148M) is the major genetic determinant of NASH / NAFLD, present in 50% of patients. Silencing SCAP activity has been proposed to benefit individuals carrying this mutation. Therefore, new therapeutic agents targeting SCAP function represent a new approach to reduce SCAP levels and treat liver diseases such as non-alcoholic fatty liver disease. Summary of the invention

[0009] The present invention is based in part on the design and generation of RNAi constructs that target the SCAP gene and reduce the expression of SCAP in hepatocytes. Sequence-specific inhibition of SCAP expression can be used to treat or prevent conditions associated with SCAP expression, such as liver-related diseases, such as simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible late-stage liver scarring), or SCAP-related obesity. Therefore, in one embodiment, the present invention provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence that is complementary to a SCAP mRNA sequence. In certain embodiments, the antisense strand comprises a region having at least 15 consecutive nucleotides from an antisense sequence listed in Table 1 or Table 2.

[0010] 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 sense strand and the antisense strand are each about 15 to about 30 nucleotides in length. In some embodiments, the RNAi construct comprises at least one blunt end. In other embodiments, the RNAi construct comprises at least one nucleotide overhang. This type of 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 with 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 the 3' end of the sense strand / 5' end of the antisense strand.

[0011] The RNAi construct of the present invention may include one or more modified nucleotides, including nucleotides with modifications to a ribose ring, a core base, or a phosphodiester backbone. In certain embodiments, the RNAi construct includes one or more 2'-modified nucleotides. Such 2'-modified nucleotides may include: 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNA), ethylene glycol nucleic acids (GNA), reverse bases (e.g., reverse adenosine) or combinations thereof. In a specific embodiment, the RNAi construct includes one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides or combinations thereof. In certain embodiments, all nucleotides in the sense strand and antisense strand of the RNAi construct are modified nucleotides.

[0012] In some embodiments, the RNAi construct comprises at least one backbone modification, such as a modified internucleotide bond or internucleoside bond. In certain embodiments, the RNAi construct described herein comprises at least one phosphorothioate internucleotide bond. In a specific embodiment, the phosphorothioate internucleotide bond may be located at the 3' or 5' end of the sense strand and / or antisense strand.

[0013] In some embodiments, the antisense strand and / or sense strand of the RNAi construct of the present invention may comprise or consist of a sequence from the antisense strand and sense sequence listed in Table 1 or Table 2. In certain embodiments, the RNAi construct may be any of the duplex compounds listed in any one of Tables 1 to 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A to Figure 1 F shows the in vivo effect of SCAP siRNA molecules in mice with the amylin (AMLN) model; (A) shows the expression of liver SCAP mRNA; (B) shows the end-stage liver weight / body weight ratio; (C) shows liver triglycerides; (D) shows serum PCSK9 levels; (E) shows the pathological readout of liver fibrosis; (F) shows aSMA staining as a marker of hepatic stellate cell activation.

[0015] Figure 2 A to Figure 2 F shows the in vivo effects of SCAP siRNA molecules in mice using the ALIOS model; (A) shows the expression of liver SCAP mRNA; (B) shows the end-stage liver weight / body weight ratio; (C) shows liver triglycerides; (D) shows serum PCSK9 levels; (E) shows the pathological readout of liver fibrosis; (F) shows aSMA staining as a marker of hepatic stellate cell activation. DETAILED DESCRIPTION

[0016] The present invention relates to compositions and methods for regulating the expression of SREBP cleavage activation protein (SCAP) gene. In some embodiments, the gene may be in a cell or subject, such as a mammal (e.g., human). In some embodiments, the composition of the present invention comprises 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 steatohepatitis (NASH), cirrhosis (irreversible advanced liver scarring), or SCAP-related obesity.

[0017] NASH / NAFLD patient populations exhibit increased expression and transcriptional activity of SREBP1c and its target genes (Higuchi et al. (2008) Hepatol Res 38, 1122-1129). Using mouse genetics and siRNA-mediated silencing, studies have shown that liver-specific ablation of SCAP activity significantly reduces liver TG content in wild-type Ob / Ob mice and hamsters fed a high-fat diet. This was accompanied by a decrease in VLDL secretion and a decrease in plasma TG levels after SCAP silencing. However, body weight, insulin and glucose levels remained unchanged (Moon et al. (2012) Cell Metab 15, 240-246). Recently, published results have shown that siRNA silencing of SCAP significantly reduced TG levels in mice and dyslipidemic macaques (Jensen et al. (2016) J Lipid Res 57, 2150-2162; Murphy et al. (2017) Metabolism 71, 202-212). Based on these published reports, we hypothesized that administration of siSCAP in NASH patients would reduce hepatic steatosis and prevent further progression of fibrosis.

[0018] RNA interference (RNAi) is the process of introducing exogenous RNA into cells, resulting in specific degradation of the mRNA encoding the targeted protein, resulting in decreased protein expression. Advances in both RNAi technology and liver delivery, as well as the increasing positive results of other RNAi-based therapies, indicate that RNAi is an effective means of therapeutically treating NAFLD by directly targeting SCAP. The inhibitory effects of these sequences were confirmed by screening Hep3B cells. We then used C57B16 mice to demonstrate that treatment with SCAP siRNA reduced SCAP expression in mice.

[0019] As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, is capable of downregulating the expression of a target gene (e.g., SCAP) using an RNA interference mechanism. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., a messenger RNA or mRNA molecule) in a sequence-specific manner, such as through an RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of continuous nucleotides that are fully complementary to each other so as to hybridize to form a duplex region. "Hybridize" or "hybridization" refers to the pairing of complementary polynucleotides, typically by hydrogen bonding (e.g., Watson-Crick hydrogen bonding, Hoogsteen hydrogen bonding, or reverse Hoogsteen hydrogen bonding) between complementary bases in two polynucleotides. A strand 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." A "sense strand" refers to a strand comprising a region that is substantially complementary to a region of an antisense strand. In some embodiments, the sense strand may comprise a region having a sequence that is substantially identical to the target sequence.

[0020] In some embodiments, the present invention provides RNAi constructs against SCAP. In some embodiments, the present invention includes RNAi constructs containing any sequence found in Table 1 or Table 2.

[0021] Double-stranded RNA molecules may 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" includes any such modifications employed in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.).

[0022] As used herein, a first sequence is "complementary" to a second sequence if a polynucleotide comprising a first sequence can hybridize to a polynucleotide comprising a second sequence to form a duplex region under certain conditions (e.g., physiological conditions). Other such conditions may include moderate or stringent hybridization conditions known to those skilled in the art. If a polynucleotide comprising a first sequence base pair is paired with a polynucleotide comprising a second sequence over the entire length of one or both nucleotide sequences and 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 sequence or target sequence by the total length of the first sequence. A sequence may also be said to be substantially complementary to another sequence if there are no more than 5, 4, 3, 2, or 1 mismatches over a 30 base pair duplex region when the two sequences are hybridized. Generally, if any nucleotide overhangs as defined herein are present, the sequence of such overhangs is not considered in determining the degree of complementarity between two sequences. For example, a sense strand of 21 nucleotides in length and an antisense strand of 21 nucleotides in length that hybridize to form a 19 base pair duplex region with a 2 nucleotide overhang at the 3' end of each strand would be considered to be fully complementary as the term is used herein.

[0023] 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, for example, having 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatch occurs in the terminal region (e.g., within 6, 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends of the strand). In one embodiment, any mismatch in the duplex region formed by the sense strand and the antisense strand occurs within 6, 5, 4, 3, 2, or 1 nucleotides at the 5' end of the antisense strand.

[0024] In certain embodiments, the sense strand and antisense strand of a double-stranded RNA can be two separate molecules that hybridize to form a duplex region that is otherwise unconnected. Such double-stranded RNA molecules formed by two separate chains are referred to as "small interfering RNA" or "short interfering RNA" (siRNA). Therefore, in some embodiments, the RNAi constructs of the present invention include siRNA.

[0025] If the two substantially complementary strands of a dsRNA are composed of separate RNA molecules, these molecules need not but can be covalently linked. If the two strands are covalently linked to form a double-stranded structure by means other than an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the corresponding other strand, the linking structure is referred to as a "joint". The RNA strands 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 strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the RNAi construct may also include one or more nucleotide overhangs.

[0026] In other embodiments, the sense strand and antisense strand that hybridize to form a duplex region can be a part of a single RNA molecule, that is, the sense strand and antisense strand are a part of the self-complementary region of a single RNA molecule. In this 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 via a continuous sequence of unpaired nucleotides, which will form a loop region. The loop region typically has enough length to allow the RNA molecule to fold back onto itself, so that the antisense strand can be base-paired with the sense strand to form a duplex region or a 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. This type of RNA molecule with at least partially self-complementary regions is referred to as "short hairpin RNA" (shRNA). In certain embodiments, the loop region can include at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In certain embodiments, the loop region can have 10, 9, 8, 7, 6, 5, 4, 3, 2, or less unpaired nucleotides. In certain embodiments, RNAi constructs of the present invention comprise 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.

[0027] In some embodiments, the RNAi construct of the present invention 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, "SCAPmRNA sequence" refers to any messenger RNA sequence encoding a SCAP protein, including splice variants, including SCAP protein variants or isoforms from any species (e.g., mouse, rat, non-human primate, human).

[0028] 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 in the form of 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 of the present invention 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 the NCBI reference sequence that may be derived from human SCAP (NM_012235) or mouse SCAP (NM_001001144).

[0029] The region of the antisense strand may be substantially complementary or completely complementary to at least 15 consecutive nucleotides of the SCAP mRNA sequence. In some embodiments, the target region of the SCAP mRNA sequence 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 consecutive nucleotides, or from about 19 to about 21 consecutive nucleotides. In certain embodiments, the region of the antisense strand comprising a sequence substantially or completely complementary to the SCAP mRNA sequence may, in some embodiments, comprise at least 15 consecutive nucleotides from the antisense sequences listed in Table 1 or Table 2. In other embodiments, the antisense sequence comprises at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides from the antisense sequences listed in Table 1 or Table 2. In some embodiments, the sense sequence and / or antisense sequence comprises at least 15 nucleotides from a sequence listed in Table 1 or Table 2 and has no more than 1, 2, or 3 nucleotide mismatches.

[0030] The sense strand of RNAi constructs typically comprises a sequence that is fully complementary to the sequence of the antisense strand, so that the two chains hybridize under physiological conditions and form a duplex region. "Duplex region" refers to a region in two complementary or substantially complementary polynucleotides, which interact by Watson-Crick base pairing or other hydrogen bonding to form base pairs, thereby forming a duplex between two polynucleotides. The duplex region of RNAi constructs should have enough length to allow RNAi constructs to enter RNA interference pathways, such as by engaging Dicer enzymes and / or RISC complexes. 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 bases, 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.

[0031] In some embodiments, the RNAi constructs of the invention contain a duplex region of about 24 to about 30 nucleotides that interacts with a target RNA sequence (e.g., a SCAP target mRNA sequence) to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells can be broken down into siRNAs using a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. [Genes and Development] 15:485). Dicer (ribonuclease-III-like enzyme) processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (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 direct 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).

[0032] For the embodiment that the sense strand and the antisense strand are two independent molecules (for example, the RNAi construct includes siRNA), 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 chains may be longer than the duplex region and have one or more unpaired nucleotides or mispairings on the side of the duplex region. Therefore, in some embodiments, the RNAi construct includes 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 chain. When the 3' end of a chain extends beyond the 5' end of another chain or when the 5' end of a chain extends beyond the 3' end of another chain, a nucleotide overhang is typically formed. 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 certain embodiments, the nucleotide overhang includes 1, 2, 3, 4, 5 or 6 nucleotides. In a specific embodiment, the nucleotide overhang includes 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In certain embodiments, the overhang comprises 5'-uridine-uridine-3' (5'-UU-3') dinucleotides. In such embodiments, the UU dinucleotides can comprise ribonucleotides or modified nucleotides, such as 2'-modified nucleotides. In other embodiments, the overhang comprises 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotides.

[0033] 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.

[0034] RNAi constructs can include single nucleotide overhangs at one end of a double-stranded RNA molecule, and include flat ends at the other end." Flat ends" 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 nucleotide overhangs at the 3' end of the sense strand, and includes flat ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct includes nucleotide overhangs at the 3' end of the antisense strand, and includes flat ends at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct includes flat ends at both ends of the double-stranded RNA molecule. In such embodiments, 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 (i.e., the molecule is double-stranded over its entire length).

[0035] The length of the sense strand and the antisense strand can be independently about 15 to about 30 nucleotides, about 18 to about 28 nucleotides, about 19 to about 27 nucleotides, about 19 to about 25 nucleotides, about 19 to about 23 nucleotides, about 21 to about 25 nucleotides, or about 21 to about 23 nucleotides. In certain embodiments, the length of the sense strand and the antisense strand is about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides. In some embodiments, the sense strand and the antisense strand have the same length, but form a duplex region shorter than these chains, so that the RNAi construct has two nucleotide overhangs. 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, and (iii) a nucleotide overhang having 2 unpaired nucleotides at 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 having a length of 21 base pairs, and (iii) a nucleotide overhang having 2 unpaired nucleotides at 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 flat-ended and comprises (i) a sense strand and an antisense strand (wherein the length of each strand is 21 nucleotides) and (ii) a duplex region having a length of 21 base pairs. In another such embodiment, the RNAi construct is flat-ended and comprises (i) a sense strand and an antisense strand (wherein the length of each strand is 23 nucleotides) and (ii) a duplex region having a length of 23 base pairs.

[0036] In other embodiments, the sense strand or the antisense strand is longer than the other strand and the two strands form a duplex region having a length equal to that of the shorter strand, such that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand 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 of 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 of 2 unpaired nucleotides at the 3' end of the antisense strand.

[0037] The antisense strand of the RNAi construct of the present invention may comprise the sequence of any one of the antisense sequences listed in Table 1 or Table 2, or the sequence of nucleotides 1-19 or 1-21 of any of these antisense sequences. Each antisense sequence listed in Table 1 and Table 2 comprises a sequence of 19 consecutive nucleotides (the first 19 nucleotides counted from the 5' end) complementary to the SCAP mRNA sequence, plus an overhang sequence of two nucleotides. Thus, in some embodiments, the antisense strand comprises a nucleotide sequence, such as nucleotides 1-19 of any one of the even-numbered sequences SEQ ID NO: 2-160, 162-320, 322-462, or 464-604. In some embodiments, the sense strand comprises a nucleotide sequence, such as nucleotides 1-19 of any one of the odd-numbered sequences SEQ ID NO: 1-159, 161-319, 321-461, or 463-603. In a specific embodiment, the antisense sequence has SEQ ID NO: 82. In a specific embodiment, the antisense sequence has SEQ ID NO:242. In a specific embodiment, the antisense sequence has SEQ ID NO:84. In a specific embodiment, the antisense sequence has SEQ ID NO:244. In a specific embodiment, the antisense sequence has SEQ ID NO:86. In a specific embodiment, the antisense sequence has SEQ ID NO:246. In a specific embodiment, the antisense sequence has SEQ ID NO:88. In a specific embodiment, the antisense sequence has SEQ ID NO:248. In a specific embodiment, the antisense sequence has SEQ ID NO:90. In a specific embodiment, the antisense sequence has SEQ ID NO:250.

[0038] Modified nucleotides

[0039] The RNAi construct of the present invention may include one or more modified nucleotides. "Modified nucleotides" refer to nucleotides with one or more chemical modifications to nucleosides, nucleobases, pentose rings, or phosphate groups. As used herein, modified nucleotides do not include: ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate; and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, the RNAi construct may include a combination of modified nucleotides, ribonucleotides, and deoxyribonucleotides. Incorporating modified nucleotides into one or both chains of a double-stranded RNA molecule can, for example, improve the in vivo stability of RNA molecules by reducing the sensitivity of the molecule to nucleases and other degradation processes. The effectiveness of RNAi constructs in reducing target gene expression can also be enhanced by the incorporation of modified nucleotides.

[0040] In certain embodiments, the modified nucleotide has a modification of ribose. These sugar modifications may include modifications at the 2' and / or 5' positions of the pentose ring, and bicyclic sugar modifications. 2'-modified nucleotides refer to nucleotides with a pentose ring having 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'-fluoro, 2'-O-methyl (OCH3), 2'-O-methoxyethyl (O-(CH2)2OCH3), 2'-OCF3, 2'-O(CH2)2SCH3, 2'-O-aminoalkyl, 2'-amino (e.g., NH2), 2'-O-ethylamine, and 2'-azido. Modifications at the 5' position of the pentose ring include, but are not limited to: 5'-methyl (R or S); 5'-vinyl and 5'-methoxy.

[0041] "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 obtain a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification is included in the bridge between the 4' and 2' carbons of the pentose ring. Nucleotides comprising sugar moieties with bicyclic sugar modifications are referred to herein as bicyclic nucleic acids or BNAs. 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 (also known as locked nucleic acids or 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); methylenethio (4'-CH2-S-2') BNA; methyleneamino (4'-CH2-N(R)-2') BNA; methylcarbocycle (4'-CH2-CH(CH3)-2') BNA; propylenecarbocycle (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 of the invention are described in U.S. Pat. No. 9,181,551, U.S. Pat. Pub. No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, all of which are hereby incorporated by reference in their entirety.

[0042] 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), ethylene glycol nucleic acids, or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In a specific embodiment, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0043] Both the sense strand and the antisense strand of the RNAi construct can include one or more modified nucleotides. For example, in some embodiments, the sense strand includes 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 certain embodiments, the antisense strand includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides can be 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides or combinations thereof.

[0044] In some embodiments, in the sense strand, the antisense strand, or both strands, all pyrimidine nucleotides in front of the adenosine nucleotide are modified nucleotides. For example, in the case where the sequence 5'-CA-3' or 5'-UA-3' appears in either strand, the cytidine nucleotide and the uridine nucleotide 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 in the antisense strand, the 5' nucleotides that appear in all sequences 5'-CA-3' or 5'-UA-3' 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.

[0045] In embodiments where the RNAi construct comprises a nucleotide overhang, the nucleotide in the overhang can 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.

[0046] The RNAi construct of the present invention may also include one or more modified internucleotide bonds. As used herein, the term "modified internucleotide bond" refers to a bond between nucleotides other than 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, an aminophosphoramidate (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 non-phosphorus-containing bonds include, but are not limited to, morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane linkages (-O-Si(H)2-O-); sulfide, sulfoxide, and sulfone linkages; formyl and thioformyl linkages; olefin-containing backbones; sulfamate backbones; methylene azomethine (-CH2-N(CH3)-O-CH2-) and methylene hydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and other linkages with mixed N, O, S, and CH2 components. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) used to form peptide nucleic acids or PNAs, as 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 employed in the RNAi constructs of the invention are described in U.S. Pat. No. 6,693,187, U.S. Pat. No. 9,181,551, U.S. Pat. Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, which are hereby incorporated by reference in their entirety.

[0047] In certain embodiments, RNAi constructs include one or more phosphorothioate internucleotide bonds. Phosphorothioate internucleotide bonds may be present in sense strand, antisense strand or both strands of RNAi constructs. For example, in some embodiments, sense strand includes 1,2,3,4,5,6,7,8 or more phosphorothioate internucleotide bonds. In other embodiments, antisense strand includes 1,2,3,4,5,6,7,8 or more phosphorothioate internucleotide bonds. In still other embodiments, both strands include 1,2,3,4,5,6,7,8 or more phosphorothioate internucleotide bonds. RNAi constructs may include one or more phosphorothioate internucleotide bonds at 3'-ends, 5'-ends or 3'-and 5'-ends of sense strand, antisense strand or both strands. For example, in certain embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) continuous phosphorothioate internucleotide 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 internucleotide bonds at the 5' end of the sense strand, antisense strand or two chains. In one embodiment, the RNAi construct comprises a single phosphorothioate internucleotide bond at the 3' end of the sense strand, and comprises a single phosphorothioate internucleotide bond at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises two continuous phosphorothioate internucleotide bonds (i.e., phosphorothioate internucleotide bonds 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 internucleotide bonds at the 3' and 5' ends of the antisense strand. In another embodiment, the RNAi construct includes two continuous phosphorothioate internucleotide bonds at the 3' and 5' ends of the antisense strand, and includes two continuous phosphorothioate internucleotide bonds at the 5' end of the sense strand. In still another embodiment, the RNAi construct includes two continuous phosphorothioate internucleotide bonds at the 3' and 5' ends of the antisense strand, and includes two continuous phosphorothioate internucleotide bonds at the 3' and 5' ends of the sense strand (that is, the phosphorothioate internucleotide bonds at the first and second nucleotides of the 5' and 3' ends of the antisense strand and the phosphorothioate internucleotide bonds at the first and second nucleotides of the 5' and 3' ends of the sense strand). In any embodiment in which one or both chains include one or more phosphorothioate internucleotide bonds, the remaining internucleotide bonds in each chain can be natural 3' to 5' phosphodiester bonds. For example, in some embodiments, each internucleotide bond of the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide bond is phosphorothioate.

[0048] In the embodiment where the RNAi construct comprises a nucleotide overhang, two or more unpaired nucleotides in the overhang can be connected via a phosphorothioate nucleotide internucleotide bond. In certain embodiments, all unpaired nucleotides in the nucleotide overhang at the 3' end of antisense strand and / or sense strand are connected via a phosphorothioate nucleotide internucleotide bond. In other embodiments, all unpaired nucleotides in the nucleotide overhang at the 5' end of antisense strand and / or sense strand are connected via a phosphorothioate nucleotide internucleotide bond. In still other embodiments, all unpaired nucleotides in any nucleotide overhang are connected via a phosphorothioate nucleotide internucleotide bond.

[0049] 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, including but not limited to: universal bases (universal bases). base), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8- Hydroxyl 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, and abasic residues (apurinic / apyrimidinic residues lacking a purine or lacking a pyrimidine base and thus lacking a nucleobase at the 1 position of the ribose sugar), and inverted nucleotides (nucleotides having a 3'-3' bond, and can be inverted nucleotides of any of the above, including inverted abasic nucleotides and inverted deoxynucleotides).

[0050] In some 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, including but 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).

[0051] 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. Chern., Vol. 76: 7295-7300, 2011, 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 containing nucleotides with such substituted nucleobases.

[0052] In some embodiments of the RNAi constructs of the present invention, the 5' end of the sense strand, the antisense strand, or both the antisense strand and the sense strand comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to a terminal phosphate group comprising an unmodified phosphate (-OP=O)(OH)OH) and a modified phosphate. The modified phosphate includes a phosphate in which one or more O and OH groups are replaced by H, O, S, N (R) or an alkyl group, wherein R is H, an amino protecting group, or an unsubstituted or substituted alkyl group. Exemplary phosphate moieties include, but are not limited to: 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or unmethylated); 5'-adenosine cap or any other modified or unmodified nucleotide cap structure; 5'-monothioate (phosphorothioate); 5'-monodithioate (phosphorodithioate); 5'-α-thiotriphosphate; 5'-γ-thiotriphosphate, 5'-phosphoramidate; 5'-vinyl phosphate; 5'-alkylphosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0053] The modified nucleotides that can be incorporated into the RNAi constructs of the present invention may have more than one chemical modification described herein. For example, the modified nucleotides may have modifications to ribose and modifications to nucleobases. For example, the modified nucleotides may include 2' sugar modifications (e.g., 2'-fluoro or 2'-methyl) and include modified bases (e.g., 5-methylcytosine or pseudouracil). In other embodiments, the modified nucleotides may include a sugar modification combination for the modification of 5' phosphate, which will form a modified internucleotide bond or internucleoside bond when the modified nucleotides are incorporated into the polynucleotide. For example, in some embodiments, the modified nucleotides may 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 two chains of the RNAi constructs of the present invention include a 2' modified nucleotide or a combination of BNA and thiophosphate internucleotide bonds. In certain embodiments, both the sense and antisense strands of the RNAi constructs of the invention comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide bonds. Table 2 shows exemplary RNAi constructs comprising modified nucleotides and internucleotide bonds.

[0054] Function of RNAi constructs

[0055] Preferably, the RNAi constructs of the present invention reduce or inhibit the expression of SCAP in cells, particularly hepatocytes. Thus, in one embodiment, the present invention provides a method for reducing 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 of the present invention can be determined relative to SCAP expression in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, the reduction of SCAP expression is assessed by: (a) measuring the amount or level of SCAP mRNA in hepatocytes treated with an RNAi construct of the invention; (b) measuring the amount or level of SCAP mRNA in hepatocytes treated with a control RNAi construct (e.g., an RNAi construct directed to an RNA molecule not expressed in hepatocytes, or an RNAi construct having a nonsense sequence or a missense sequence) or treated with no 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 transcription polymerase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

[0056] In other embodiments, the reduction of SCAP expression is assessed by: (a) measuring the amount or level of SCAP protein in hepatocytes treated with an RNAi construct of the invention; (b) measuring the amount or level of SCAP protein in hepatocytes treated with a control RNAi (e.g., an RNAi construct directed to an RNA molecule not expressed in hepatocytes, or an RNAi construct having a nonsense or missense sequence); 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). Methods for measuring SCAP protein levels are known to those skilled in the art and include Western blots, immunoassays (e.g., ELISA), and flow cytometry. Example 3 describes an exemplary method for measuring SCAP mRNA using RNA FISH. Any method capable of measuring SCAP mRNA or protein can be used to assess the efficacy of the RNAi constructs of the invention.

[0057] In some embodiments, the method of assessing the expression level of SCAP is performed in vitro in cells that naturally express SCAP (e.g., hepatocytes) or cells that have been engineered to express SCAP. In certain embodiments, the methods are performed in vitro in hepatocytes. Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., hepatocytes 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.

[0058] In other embodiments, the method of assessing SCAP expression levels is performed in vivo. The RNAi construct and any control RNAi constructs can be administered to an animal (e.g., a rodent or non-human primate), and SCAP mRNA or protein levels can be assessed in liver tissue harvested from the animal after treatment. Alternatively or additionally, biomarkers or functional phenotypes associated with SCAP expression can be assessed in the treated animals.

[0059] In certain embodiments, the RNAi constructs of the invention reduce the expression of SCAP in hepatocytes by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%. In some embodiments, the RNAi constructs of the invention reduce the expression of SCAP in hepatocytes by 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 of the invention reduce the expression of SCAP in hepatocytes 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 using any of the methods described herein and other methods known in the art. For example, in certain embodiments, the RNAi constructs of the invention inhibit the expression of SCAP in Hep3B cells (including wild-type SCAP) by at least 45% in vitro, as described in Examples 2 and 4. In related embodiments, the RNAi constructs of the invention inhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% of SCAP expression in Hep3B cells in vitro, as described in Examples 2 and 4. In other embodiments, the RNAi constructs of the invention inhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96% or at least 98% of SCAP expression in Hep3B cells in vitro, as described in Examples 2 and 4. In certain embodiments, the RNAi constructs of the invention inhibit at least 45% of SCAP expression in C57B16 mouse liver, as described in the examples. In related embodiments, the RNAi constructs of the invention inhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75% of SCAP expression in C57B16 mouse liver, as described in the examples. In other embodiments, the RNAi constructs of the invention inhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of SCAP expression in C57B16 mouse liver, as described in the Examples. Reduction of SCAP can be measured using a variety of techniques, including RNA FISH or droplet digital PCR as described in Examples 2 and 4, or in vivo studies as described in Examples 3, 5, 6, 7, and 8.

[0060] In some embodiments, IC50 values ​​are calculated to assess the efficacy of the RNAi constructs of the present invention in inhibiting the expression of SCAP in hepatocytes. An "IC50 value" is the dose / concentration required to achieve 50% inhibition of a biological or biochemical function. The IC50 value of any particular substance or antagonist can be determined by plotting a dose-response curve and examining the effects of different concentrations of the substance or antagonist on expression levels or functional activity in any assay. The IC50 value of a given antagonist or substance can be calculated by determining the concentration required to inhibit half of the maximum biological response or native expression level. Thus, the IC50 value of any RNAi construct can be calculated by determining the concentration of the RNAi construct required to inhibit half of the native SCAP expression level in hepatocytes (e.g., the expression level of control SCAP in hepatocytes) in any assay (e.g., an immunoassay or RNA FISH assay or a droplet digital PCR assay described in each example). The RNAi constructs of the present invention can inhibit the expression of SCAP in hepatocytes (e.g., Hep3B cells) with an IC50 of less than about 100 nM. For example, the RNAi construct inhibits the expression of SCAP in hepatocytes with an IC50 of about 0.001 nM to about 100 nM, about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits the expression of SCAP in hepatocytes (e.g., Hep3B cells) with an IC50 of about 1 nM to about 10 nM. In certain embodiments, the RNAi construct inhibits the expression of SCAP in hepatocytes (e.g., Hep3B cells) with an IC50 of about 0.1 nM to about 5 nM. The RNAi constructs of the present invention can inhibit the expression of SCAP in hepatocytes (e.g., Hep3B cells) with an IC50 of less than about 20 nM. For example, the RNAi construct inhibits the expression of SCAP in hepatocytes with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits the expression of SCAP in hepatocytes (e.g., Hep3B cells) with an IC50 of about 1 nM to about 10 nM.

[0061] In some embodiments, the RNAi constructs of the invention can have prolonged silencing of SCAP in vivo, such as in ob / ob mice as described in Example 8. In some embodiments, the RNAi constructs of the invention can silence at least 50%, at least 70%, or at least 80% of SCAP expression 20 days after administration of the construct in ob / ob mice, as described in Example 8. In some embodiments, the RNAi constructs of the invention can silence at least 50%, at least 60%, or at least 70% of SCAP expression 30 days after administration of the construct in ob / ob mice, as described in Example 8.

[0062] RNAi constructs of the present invention can be easily made using techniques known in the art (e.g., using conventional nucleic acid solid phase synthesis). Standard nucleotides or nucleoside precursors (e.g., phosphoramidites) can be used to assemble the polynucleotides of RNAi constructs on a suitable nucleic acid synthesizer. Automatic nucleic acid synthesizers are commercially sold by several suppliers, including DNA / RNA synthesizers from Applied Biosystems (Foster City, California), MerMade synthesizers from BioAutomation (Irving, Texas), and OligoPilot synthesizers from GE Healthcare Life Sciences (Pittsburgh, Pennsylvania).

[0063] 2' silyl protecting groups can be used in combination with acid-labile dimethoxytrityl (DMT) at the 5' position of ribonucleosides to synthesize oligonucleotides using phosphoramidite chemistry. It is known that the final deprotection conditions will not significantly degrade the RNA product. All synthesis can be performed on a large, medium or small scale in any automatic or manual synthesizer. Synthesis can also be performed in multiple well plates, columns or slides.

[0064] The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any fluoride ion source, such as salts containing fluoride ions paired with inorganic counterions (e.g., cesium fluoride and potassium fluoride), or salts containing fluoride ions paired with organic counterions (e.g., tetraalkylammonium fluorides). In the deprotection reaction, a crown ether catalyst can be used in combination with an inorganic fluoride. A preferred fluoride ion source is tetrabutylammonium fluoride, or an aminohydrofluoride (e.g., combining aqueous HF with triethylamine in a dipolar aprotic solvent such as dimethylformamide).

[0065] The choice of protecting groups used for phosphite triesters and phosphate triesters can change the stability of the triesters to fluoride. Methyl protection of phosphate triesters or phosphite triesters can stabilize the bond to fluoride ions and improve process yields.

[0066] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in the RNA with a protecting group perpendicular to the 5'-O-dimethoxytrityl protecting group (e.g., one that is stable to treatment with acid). Silyl protecting groups meet this criterion and can be easily removed in the final fluoride deprotection step, which can result in minimal RNA degradation.

[0067] In the standard phosphoramidite coupling reaction, a tetrazole catalyst can be used. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylmercaptotetrazole, and p-nitrophenyltetrazole.

[0068] As will be appreciated by those of ordinary skill in the art, other methods of synthesizing the RNAi constructs described herein will be apparent to those of skill in the art. In addition, the various synthetic steps may be performed in an alternate sequence or order to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., hydroxyl, amino, etc. present on bases), and protecting group methods (protection and deprotection) useful in synthesizing the RNAi constructs described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations VCH Press (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 editions. Custom synthesis of RNAi constructs can also be achieved from several commercial suppliers including Dharmacon, Inc. (Lafayette, Colorado), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, California).

[0069] The RNAi constructs of the present invention may include ligands. As used herein, "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 cause a biological response (e.g., triggering a signal transduction cascade, inducing receptor-mediated endocytosis) or simply physical binding. The ligand may change one or more properties attached to the double-stranded RNA molecule, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance properties of the RNA molecule.

[0070] The ligand can include a serum protein (e.g., human serum albumin, low-density lipoprotein, globulin), a cholesterol moiety, a vitamin (biotin, vitamin E, vitamin B12), a folate moiety, a steroid, a bile acid (e.g., cholic acid), a fatty acid (e.g., palmitic acid, myristic acid), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), a glycoside, a phospholipid, or an antibody or binding fragment thereof (e.g., an antibody or binding fragment thereof that targets the RNAi construct to a specific cell type, such as the liver). Other examples of ligands include dyes, intercalators (e.g., acridine), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, lipophilic molecules (e.g., adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-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., Drosophila melanogaster antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0071] In certain embodiments, the ligand has an endosomolytic property. The endosomolytic ligand promotes the dissolution of the endosomal and / or the transport of the RNAi construct of the present invention or its components from the endosome to the cytoplasm of the cell. The endosomolytic ligand can be a polycationic peptide or peptide mimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand presents its active conformation at endosomal pH. An "active" conformation is one in which the endosomolytic ligand promotes the dissolution of the endosomal and / or the transport of the RNAi construct of the present invention or its components from the endosome to the cytoplasm. 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 will undergo changes in charge or protonation as the pH changes. The endosomolytic component may be linear or branched.

[0072] 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 has been reported that cholesterol-conjugated oligonucleotides are more active than their unconjugated oligonucleotides (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules have been described in U.S. Patent Nos. 7,851,615; 7,745,608; and 7,833,992, which are hereby incorporated by reference in their entirety. In another embodiment, the ligand comprises a folic acid moiety. The polynucleotide conjugated to the folic acid moiety can be taken up by cells via a receptor-mediated endocytic pathway. Such folic acid-polynucleotide conjugates are described in U.S. Patent No. 8,188,247, which is hereby incorporated by reference in its entirety.

[0073] Given that SCAP is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to deliver RNAi constructs specifically to these 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 ligand may comprise an antigen binding protein (e.g., an antibody or binding fragment thereof (e.g., Fab, scFv)) that specifically binds to a receptor expressed on a liver cell.

[0074] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound consisting of one or more monosaccharide units (which may be linear, branched or cyclic) having at least 6 carbon atoms, wherein an oxygen, nitrogen or sulfur atom is bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides (such as starches, glycogen, cellulose, and polysaccharide gums). In some embodiments, the carbohydrate incorporated into the ligand is selected from a monosaccharide of a pentose, hexose, or heptose and a disaccharide and trisaccharide comprising such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0075] In some embodiments, the ligand includes 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 includes glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand includes glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand includes galactose, galactosamine, or N-acetyl-galactosamine. In a particular embodiment, the ligand includes N-acetyl-galactosamine. The ligand including glucose, galactose, and N-acetyl-galactosamine (GalNAc) is particularly effective in targeting compounds to hepatocytes. See, for example, D'Souza and Devarajan, J.Control Release, Vol. 203: 126-139, 2015. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs of the invention are described in U.S. Patent Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Publication No. 20030130186; and WIPO Publication No. WO 2013166155, which are hereby incorporated by reference in their entireties.

[0076] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety comprising two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains, which are composed of carbohydrates that can bind to two or more different molecules or two or more different sites on the same molecule. The valence of the carbohydrate moiety represents the number of separate binding domains within the carbohydrate moiety. For example, the terms "monovalent", "divalent", "trivalent" and "tetravalent" with respect to the carbohydrate moiety refer to carbohydrate moieties having one, two, three and four binding domains, respectively. The multivalent carbohydrate moiety may comprise: a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety is divalent, trivalent or tetravalent. In such embodiments, the multivalent carbohydrate moiety can 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 trivalent and tetravalent GalNAc-containing ligands for incorporation into RNAi constructs of the present invention are described in detail below.

[0077] The ligand can be directly or indirectly attached or conjugated to the RNA molecule of RNAi construct.For example, in some embodiments, the ligand is directly covalently attached to the sense strand or antisense strand of RNAi construct.In other embodiments, the ligand is covalently attached to the sense strand or antisense strand of 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 RNAi construct of the present invention.With the conjugation or attachment of purine core base or its derivative, it can occur in any position including the atoms inside and outside the ring.In certain embodiments, the 2 position, 6 position, 7 position or 8 position of purine core base are attached to the ligand.With the conjugation or attachment of pyrimidine core base or its derivative can also occur in any position.In certain embodiments, the 2 position, 5 position and 6 position of pyrimidine core base can be attached to the ligand.With the conjugation or attachment of the sugar moiety of nucleotide can occur at any carbon atom. The example of the carbon atom that can be attached to the sugar part of the part includes 2', 3' and 5' carbon atom. The 1' position can also be attached to the part, as in basic residues. The internucleotide bond can also support the attachment of the part. With regard to phosphorus-containing bonds (for example, phosphodiester, thiophosphate, dithiophosphate, phosphoramidate, etc.), the part can be directly attached to the phosphorus atom or attached to the O, N or S atom combined with the phosphorus atom. With regard to the internucleoside bond (for example, PNA) containing amine or amide, the part can be attached to the nitrogen atom of amine or amide or attached to the adjacent carbon atom.

[0078] In certain embodiments, the ligand can be attached to the 3' end or 5' end of any one of the sense strands or antisense strands. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In some such embodiments, the ligand is attached to 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 certain embodiments, the ligand is attached to the 2' position of the sugar of the 3'-terminal nucleotide of the sense strand.

[0079] In certain embodiments, the ligand is attached to the sense strand or antisense strand via a joint. "Joint" is an atom or group that makes the ligand covalently attached to the polynucleotide component of the RNAi construct. The joint can be a length of about 1 to about 30 atoms, a length of about 2 to about 28 atoms, a length of about 3 to about 26 atoms, a length of about 4 to about 24 atoms, a length of about 6 to about 20 atoms, a length of about 7 to about 20 atoms, a length of about 8 to about 20 atoms, a length of about 8 to about 18 atoms, a length of about 10 to about 18 atoms, and a length of about 12 to about 18 atoms. In certain embodiments, the joint may include a bifunctional linking portion, which generally includes an alkyl portion with a functional group. One of the functional groups is selected to be combined with the compound of interest (such as the sense strand or antisense strand of the RNAi construct), and another functional group is selected to be substantially combined with any selected group (such as a ligand 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 employed in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups, and nucleophiles for reacting with electrophilic groups. In some embodiments, the bifunctional linking moiety includes amino, hydroxyl, carboxylic acid, thiol, unsaturated bonds (e.g., double bonds or triple bonds), and the like.

[0080] Linkers that can be used to attach ligands to the sense or antisense strands in the RNAi constructs of the invention 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-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0081] In certain embodiments, the joint is cleavable. A cleavable joint is a joint that is fully stable outside the cell, but is cleaved to release the two parts of the joint that are kept together after entering the target cell. In certain embodiments, a cleavable joint is in the target cell or under a first reference condition (for example, the condition can be selected to simulate or represent the intracellular condition), than in the blood of the experimenter or under a second reference condition (for example, the condition can be selected to simulate or represent the condition found in blood or serum) at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or higher or at least 100 times cleaved.

[0082] Cleaving joints are susceptible to the effects of cleavage agents, such as pH, redox potential or the presence of degradable molecules. Typically, cleavage agents are more prevalent or found at higher levels or higher activity inside cells than in serum or blood. Examples of such degradants include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidases or reductases or reducing agents (such as thiols) present in cells that can degrade oxidative reducible cleavable joints by reduction; esterases; endosomes or reagents that can form an acidic environment, for example, those that result in a pH of 5 or less; enzymes that hydrolyze or degrade acid cleavable joints by acting as generalized acids, peptidases (which can be substrate specific) and phosphatases.

[0083] The cleavable linker may comprise a pH-sensitive moiety. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, in the range of about 7.1-7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have a more acidic pH around 5.0. Some linkers will have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand inside the cell or into a desired cell compartment.

[0084] The joint can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the joint can depend on the cell to be targeted. For example, the part of the liver can be connected to the RNA molecule via a joint including an ester group. Hepatocytes are rich in esterases, so compared with cell types that are not rich in esterases, the joint is more efficiently cleaved in hepatocytes. Other types of cells that are rich in esterases include cells of the lung, renal cortex and testis. When targeting is rich in cells (such as hepatocytes and synoviocytes) that are peptidase, a joint containing a peptide bond can be used.

[0085] Generally, the suitability of a candidate cleavable joint can be assessed by testing the ability of a candidate joint cracking a degradation agent (or condition). It will also be desirable to test the ability of a candidate cleavable joint to resist cracking in blood or when contacting with other non-target tissues. Therefore, the relative cracking sensitivity between the first condition and the second condition can be determined, wherein the first condition is selected as the indication of cracking in the target cell and the second condition is selected as the indication of cracking in other tissues or biofluids (e.g., blood or serum). It can be assessed in a cell-free system, cell, cell culture, organ or tissue culture or whole animal. It may be useful to conduct a preliminary assessment under cell-free or culture conditions, and to be confirmed by further assessment in whole animal. In certain embodiments, the cracking of a useful candidate joint in a cell (or under the in vitro conditions selected for simulating intracellular conditions) is at least 2, 4, 10, 20, 50, 70 or 100 times faster than in blood or serum (or under the in vitro conditions of simulating extracellular conditions).

[0086] In other embodiments, an oxidation-reducible cleavage joint is used. An oxidation-reducible cleavage joint is cleaved when reduced or oxidized. An example of a reducible cleavage group is a disulfide linking group (-SS-). In order to determine whether a candidate cleavable joint is a suitable "reducible cleavage 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, by hatching with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate observed in a cell (for example, a target cell), a candidate joint can be evaluated. It is also possible to evaluate a candidate joint under conditions selected to simulate blood or serum conditions. In a specific embodiment, a candidate joint is cleaved at most 10% in blood. In other embodiments, there is a useful candidate joint in a cell (or under conditions selected to simulate intracellular conditions in vitro) degradation, compared with in blood (or under conditions selected to simulate extracellular conditions), at least 2, 4, 10, 20, 50, 70 or 100 times faster.

[0087] In yet other embodiments, the phosphate-based cleavable linker is cleaved by a cleaving agent that degrades or hydrolyzes the phosphate group. Examples of cleaving 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)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Specific embodiments include: -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. Another specific embodiment is -OP(O)(OH)-O-. These candidate joints can be evaluated using methods similar to those described above.

[0088] In other embodiments, the linker may include an acid-cleavable group, which is a group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable group is cleaved in an acidic environment of about pH 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or is cleaved by a cleaving agent (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 linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. 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) of the ester is an aryl group, a specific embodiment is a substituted alkyl group, or a tertiary alkyl group (such as a dimethyl, pentyl, or tert-butyl group). These candidates can be evaluated using methods similar to those described above.

[0089] In other embodiments, the joint can include ester-based cleavable groups that are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene. Ester cleavable groups have the general formula -C(O)O- or -OC(O)-. These candidate joints can be evaluated using methods similar to those described above.

[0090] In a further embodiment, the linker may comprise peptide-based cleavable groups that are cleaved by enzymes such as peptidases and proteases in cells. 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 and proteins to produce peptides, and do not include the entire amide functional group. Peptide-based cleavable linkers have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0091] Other types of linkers suitable for attaching ligands to the sense or antisense strands in the RNAi constructs of the invention are known in the art and may include linkers described in U.S. Pat. Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551 (all of which are hereby incorporated by reference in their entirety).

[0092] In certain embodiments, the ligand covalently attached to the sense strand or antisense strand of the RNAi construct of the present invention comprises a GalNAc moiety, such as a multivalent GalNAc moiety. In certain 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 other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety, and is attached to the 3' end of the sense strand. In still other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety, and is attached to the 5' end of the sense strand. In certain embodiments, the GalNAc moiety is attached to the 5' end of the sense strand of the odd numbered sequence SEQ ID NO:1-159, 161-319, 321-461 or 463-603.

[0093] In some embodiments, the RNAi construct of the present invention can be delivered to a target cell or tissue by administering a vector that encodes and controls the intracellular expression of the RNAi construct. A "vector" (also referred to herein as an "expression vector") is a composition of matter that can be used to deliver a target nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic 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 a living cell, or it can be prepared by synthesis.

[0094] Typically, the vector for expressing the RNAi construct of the present invention will contain one or more promoters operably linked to the sequence encoding the RNAi construct. As used herein, the phrase "operably linked" or "under transcriptional control" means that the promoter is in the correct position and orientation relative to the polynucleotide sequence to control the transcription initiation of RNA polymerase and the expression of the polynucleotide sequence. "Promoter" refers to a sequence required for specific transcription of a gene sequence that is recognized by the synthetic machinery of the cell or introduced by the synthetic machinery. Suitable promoters include, but are not limited to, RNA pol I, polII, HI or U6 RNA pol III, and viral promoters (e.g., human cytomegalovirus (CMV) early gene promoter, SV40 early promoter, and Rous sarcoma virus long terminal repeat sequence). In some embodiments, HI or U6 RNA pol III promoters are preferred. The promoter can 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 promoters regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-PD1-thiogalactopyranoside (IPTG).

[0095] In some embodiments in which RNAi constructs include siRNA, two independent chains (sense strand and antisense strand) can be expressed from a single vector or two independent vectors. For example, in one embodiment, 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 this type of embodiment, for example, by infection or transfection, the first vector and the second vector are introduced into the target cell together, so that the sense strand and the antisense strand will hybridize in the cell to form the siRNA molecule once transcribed. In another embodiment, the sense strand and the antisense strand are transcribed from two independent promoters in a single vector. In some of these embodiments, the sequence encoding the sense strand is operably connected to the first promoter, and the sequence encoding the antisense strand is 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.

[0096] In other embodiments where the RNAi construct comprises an 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 via a linker polynucleotide sequence, thereby generating a stem-loop structure of the shRNA after transcription.

[0097] In some embodiments, the vector encoding the RNAi construct of the present invention is a viral vector. Various viral vector systems suitable for expressing the RNAi constructs described herein include, but are not limited to, adenoviral vectors, retroviral vectors (e.g., lentiviral vectors, Maloney murine leukemia virus), adeno-associated viral vectors; herpes simplex virus vectors; SV 40 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).

[0098] Various vectors suitable for use in the present invention, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into vectors, and methods for delivering vectors to target cells are within the scope of those skilled in the art. See, for example, Dornburg, Gene Therap., Vol. 2: 301-310, 1995; Eglitis, Biotechniques, Vol. 6: 608-614, 1988; Miller, Hum Gene Therap., Vol. 1: 5-14, 1990; Andseson, 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. 99:5515-5520, 2002. Proceedings of the National Academy of Sciences USA, Vol. 99: 6047-6052, 2002, all of which are hereby incorporated by reference in their entirety.

[0099] The present invention also includes pharmaceutical compositions and formulations comprising the RNAi constructs described herein and a pharmaceutically acceptable carrier, excipient and / or diluent. Such compositions and formulations can be used to reduce the expression of SCAP in a subject in need thereof. In the case of clinical applications, pharmaceutical compositions and formulations will be prepared in a form suitable for the intended application. Typically, this will require the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0100] 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 of the present invention, they may be considered for use in therapeutic compositions. Supplementary active ingredients may also be incorporated into the composition, as long as they do not inactivate the carrier or RNAi construct of the composition.

[0101] The composition and method for preparing the pharmaceutical composition depend on some conditions, including but not limited to: route of administration, type and degree of the disease or disorder to be treated or dosage to be administered. In some embodiments, the pharmaceutical composition is prepared based on the expected delivery route. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery forms include: intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such embodiments, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such embodiments, the pharmaceutical composition may include a targeting ligand (e.g., a ligand containing GalNAc described herein).

[0102] 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 the expression of SCAP, for example, to a level comparable to the expression of a wild-type SCAP allele in a human heterozygote.

[0103] The effective amount of RNAi construct of the present invention can be 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. In certain embodiments, the single effective dose of RNAi construct of the present invention can be about 0.1mg / kg, about 0.5mg / kg, about 1mg / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg or about 10mg / kg. The pharmaceutical composition comprising the RNAi construct of effective amount can be applied once weekly, every two weeks, monthly, quarterly or every half a year. The precise determination of a specific effective dose and the circumstances that should be considered for the frequency of administration can be based on several factors, including the size, age and general condition of the patient, the type of disorder to be treated (e.g., myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia), the RNAi construct employed, and the route of administration. An estimate of the effective dose and in vivo half-life of any particular RNAi construct of the invention can be determined using conventional methods and / or testing in appropriate animal models.

[0104] The administration of the pharmaceutical composition of the present invention can be carried out via any common route, as long as the target tissue can be reached via the route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal or intravenous), oral, nasal, buccal, intradermal, transdermal and sublingual routes, or by direct injection into liver tissue or delivery through the portal vein. In some embodiments, the pharmaceutical composition is parenteral administration. For example, in certain embodiments, the pharmaceutical composition is intravenously administered. In other embodiments, the pharmaceutical composition is subcutaneously administered.

[0105] 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 RNAi constructs of the present invention or vectors encoding such constructs. Commercially available fat emulsions suitable for delivering nucleic acids of the present invention include: II. III, Nutrilipid, and other similar lipid emulsions. The preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi construct of the present invention can be encapsulated in a liposome or can form a complex with it, particularly a cationic liposome. Alternatively, the RNAi construct of the present invention can form a complex with a lipid, particularly a cationic lipid. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC)), distearoylphosphatidylcholine), and negative (e.g., dimyristoylphosphatidylglycerol (DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems are described herein. Exemplary formulations are also disclosed in U.S. Pat. No. 5,981,505; U.S. Pat. No. 6,217,900; U.S. Pat. No. 6,383,512; U.S. Pat. No. 5,783,565; U.S. Pat. No. 7,202,227; U.S. Pat. No. 6,379,965; U.S. Pat. No. 6,127,170; U.S. Pat. No. 5,837,533; U.S. Pat. No. 6,747,014; and WO03 / 093449.

[0106] In certain embodiments, the RNAi construct of the present invention is completely encapsulated in a lipid formulation, for example, to form SPLP (stable plasmid-lipid particles), pSPLP, SNALP (stable nucleic acid lipid nanoparticles) 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 applications because they show extended circulation life after intravenous injection, and accumulate at distal sites (for example, sites physically separated from the site of application). SPLP includes "pSPLP", which includes encapsulation condensing agent-nucleic acid complexes as stated in PCT Publication No. WO00 / 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, when present in the nucleic acid-lipid particles, the nucleic acids are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed in, for example, U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96 / 40964.

[0107] 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 fluid to the extent that they are easy to inject. The preparation should be stable under the conditions of manufacture and storage, and should prevent the contamination of microorganisms (such as bacteria and fungi). Suitable solvents or dispersion media may include, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), their suitable mixtures, and vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the required particle size in the case of a dispersion, and 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, it will be more preferred to include isotonic agents (such as sugar or sodium chloride). The extended absorption of injectable compositions can be achieved by using absorption delay agents (such as aluminum monostearate and gelatin) in the composition.

[0108] Sterile injectable solutions can be prepared by incorporating the active compound in an appropriate amount into a solvent along with any other ingredients as required (e.g., as listed above) followed by filtered sterilization. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other ingredients as required, such as those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying techniques that produce powders of one or more active ingredients plus any other desired ingredients from previously sterile filtered solutions thereof.

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

[0110] For example, in terms of parenteral administration in an aqueous solution, the solution is usually appropriately buffered, and first, for example, a liquid diluent is made isotonic with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, as known to those skilled in the art, in particular according to the present disclosure, a sterile aqueous medium is used. By way of example, a single dose can be dissolved in an isotonic NaCl solution of 1 ml, or added to a subcutaneous perfusion solution of 1000 ml, or injected at the infusion site of the suggestion (see, for example, Remington's Pharmaceutical Sciences, 15th edition, 1035-1038 pages and 1570-1580 pages). In terms of human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises a sterile saline solution and an RNAi construct described herein or is composed thereof. In other embodiments, the pharmaceutical composition of the present invention comprises RNAi constructs described herein and sterile water (e.g., water for injection, WFI) or consists of it. In still other embodiments, the pharmaceutical composition of the present invention comprises RNAi constructs described herein and phosphate buffered saline (PBS) or consists of it.

[0111] In some embodiments, the pharmaceutical compositions of the present invention are packaged or stored in a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, prefilled syringes, automatic syringes, injection pumps, on-body 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 invention includes an administration device comprising a pharmaceutical composition of the present invention for use in treating or preventing one or more disorders described herein.

[0112] Methods for inhibiting SCAP expression

[0113] The present invention 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, thereby inhibiting the expression of SCAP in the cell. The contact of 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 the cell are also possible.

[0114] The present invention provides methods for reducing or inhibiting the expression of SCAP in a subject in need thereof, and methods for treating or preventing a condition, disease, or disorder associated with SCAP expression or activity. A "condition, disease, or disorder associated with SCAP expression" refers to a condition, disease, or disorder in which the level of SCAP expression is altered or in which an elevated level of SCAP expression is associated with an increased risk of developing the condition, disease, or disorder.

[0115] As described above, contact with cells can be direct or indirect. In addition, contact with cells can be achieved by targeting ligands, including any ligands described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or a trivalent GalNAc moiety, or any other ligand that directs the RNAi construct to the site of interest.

[0116] In one embodiment, contacting a cell with an RNAi construct includes "introducing or delivering the RNAi construct into a cell" by promoting or enabling uptake or absorption into the cell. The absorption or uptake of the RNAi construct can occur by independent diffusion or active cellular processes, or by utilizing an adjuvant or device. The introduction of the RNAi construct into a cell can be performed in vitro and / or in vivo. For example, with respect to in vivo introduction, the RNAi construct can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and liposome transfection. Other methods are described below and / or are known in the art.

[0117] As used herein, the term "inhibiting" is used interchangeably with "reducing," "silencing," "downregulating," "suppressing," and other similar terms, and includes any level of inhibition.

[0118] The phrase "inhibiting the expression of SCAP" is intended to mean the inhibition 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 a 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 (such as a mutant SCAP gene that causes triglyceride deposition), or a transgenic SCAP gene.

[0119] "Inhibiting the expression of the SCAP gene" includes any level of inhibition of the SCAP gene, for example, at least partial inhibition 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, SCAP protein level, or the amount or extent of triglyceride deposition). The level can be assessed in a single cell or in a cell population (including, for example, a sample derived from a subject).

[0120] 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, such as a baseline level prior to administration, or a level determined in a similar subject, cell or sample that has never been treated with a control or that has been treated with a control (e.g., a buffer-only control or an inactive agent control). In some embodiments of the methods of the present invention, 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%.

[0121] Inhibition of SCAP gene expression can be demonstrated by a reduction in the amount of mRNA expressed by a first cell or population of cells (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 of the invention, or by administering an RNAi construct of the invention to a subject in which such cells are or have been present) such that expression of the SCAP gene is inhibited as compared to a second cell or population of cells (control cells) that is substantially identical to the first cell or population of cells but has not been so treated. In a preferred embodiment, inhibition is 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:

[0122]

[0123] Alternatively, inhibition of SCAP gene expression can be assessed using parameters that are functionally related to SCAP gene expression (e.g., reduction in SCAP protein expression or SREBP pathway protein activity). SCAP gene silencing can be determined in any cell that expresses SCAP, either constitutively or genomically engineered, by any assay known in the art.

[0124] Inhibition of SCAP protein expression can be demonstrated by reducing the level of SCAP protein expressed by a cell or cell population (e.g., the level of protein expressed in a sample derived from a subject). As described above, for the assessment of mRNA inhibition, the inhibition of protein expression levels in treated cells or cell populations can be similarly expressed as a percentage of the protein level in control cells or cell populations.

[0125] 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 of the invention. For example, control cells or cell populations can be derived from a separate subject (e.g., a human or animal subject) prior to treatment of the subject with the RNAi construct.

[0126] 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. In one embodiment, the level of SCAP expression in a sample is determined by detecting a transcribed polynucleotide or portion thereof (e.g., mRNA of a SCAP gene). RNA can be extracted from cells using RNA extraction techniques, including, for example, extraction using acid phenol / guanidine isothiocyanate (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical detection methods using RNA hybridization include: nuclear run-on assay, RT-PCR, RNase protection assay (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating SCAP mRNA can be detected using the methods described in PCT / US2012 / 043584, which is hereby incorporated by reference in its entirety.

[0127] In one embodiment, a nucleic acid probe is 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. The probe can be synthesized by a person skilled in the art, or derived from an appropriate biological product. 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.

[0128] The isolated mRNA can be used for hybridization or amplification assays, including but not limited to Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method of determining mRNA levels includes contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with SCAP mRNA. In one embodiment, the mRNA is fixed to 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 a nitrocellulose membrane). In an alternative embodiment, the one or more probes are fixed to 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.

[0129] An alternative method for determining the level of SCAP expression in a sample includes, 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 (Mullis, 1987, as described in 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 88:189-193), or cDNA sequencing (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 88:189-193). USA [Proceedings of the National Academy of Sciences of the United States of America] 87: 1874-1878), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 86: 1173-1177), Q-β replicase (Lizardi et al. (1988) Bio / Technology [Biotechnology] 6: 1197), rolling circle replication (Lizardi et al., U.S. Patent 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 detection of nucleic acid molecules if such molecules are present in very low numbers. In a specific aspect of the invention, the nucleic acid molecules are detected by fluorescent quantitative RT-PCR (i.e., TaqMan TMThe expression level of SCAP can be determined by a method of measuring the expression level of SCAP using a PCR system. The expression level of SCAP mRNA can be monitored using membrane blotting (such as used in hybridization analysis such as Northern hybridization analysis, dot blot analysis, etc.), or microwells, sample tubes, gels, beads or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, the contents of which are incorporated herein by reference. The determination of the expression level of SCAP can also include the use of a nucleic acid probe in solution.

[0130] In preferred embodiments, the level of mRNA expression is assessed using a branched DNA (bDNA) assay or real-time PCR (qPCR). The use of these methods is described and exemplified in the examples provided herein.

[0131] 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 precipitation reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double direction), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0132] In some embodiments, the efficacy of the methods of the invention can be monitored by detecting or monitoring symptoms of SCAP disease, such as edema of the extremities, face, larynx, upper respiratory tract, abdomen, trunk, and genitalia, prodrome; swelling of the throat; non-pruritic rash; nausea; vomiting; or relief of abdominal pain. These symptoms can be assessed in vitro or in vivo using any method known in the art.

[0133] In some embodiments of the methods of the present invention, the RNAi construct is administered to a subject, thereby delivering the RNAi construct to a specific site within the subject. The inhibition of SCAP expression can be assessed by measuring the level or change in the level of SCAP mRNA or SCAP protein in a sample of fluid or tissue derived from a specific site of the subject. In a preferred embodiment, the site is selected from the group consisting of: liver, choroid plexus, retina, and pancreas. The site may also be a subset or subpopulation of cells from any of the aforementioned sites. The site may also include cells that express a specific type of receptor.

[0134] Methods for treating or preventing SCAP-related diseases

[0135] The present invention provides methods of treatment and prevention, comprising administering a composition comprising RNAi, or a pharmaceutical composition comprising an RNAi construct, or a vector comprising an RNAi construct of the present invention to a subject suffering from or susceptible to developing a SCAP-related disease, disorder, and / or condition. Non-limiting examples of SCAP-related diseases include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, inflammation of the liver, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia, or non-alcoholic fatty liver disease (NAFLD). In one embodiment, the SCAP-related disease is NAFLD. In another embodiment, the SCAP-related disease is NASH. In another embodiment, the SCAP-related disease is fatty liver (steatosis). In another embodiment, the SCAP-related disease is insulin resistance. In another embodiment, the SCAP-related disease is not insulin resistance. In some embodiments, SCAP RNAi can be used to treat hepatocellular carcinoma. Increased SREBP activity has been documented in human HCC samples and there is evidence for a causal role in hepatocellular carcinoma (HCC) growth. SCAP RNAi (e.g., siRNA) treatment in rodent models of HCC (e.g., xenografts of HCC cells or hepatic expression of oncogenes) can result in a reduction in liver tumor burden (i.e., tumor volume).

[0136] In certain embodiments, the present invention provides a method of reducing SCAP expression 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 can be used interchangeably with the term "subject". Preferably, the expression level of SCAP in hepatocytes in the patient is reduced after administration of the RNAi construct compared to the expression level of SCAP in the patient who did not receive the RNAi construct.

[0137] The methods of the present invention can be used to treat subjects with SCAP-related diseases, such as subjects who will benefit from reduced SCAP gene expression and / or SCAP protein production. In one aspect, the present invention provides methods for reducing the expression level of the SREBP cleavage activation protein (SCAP) gene in subjects with non-alcoholic fatty liver disease (NAFLD). In another aspect, the present invention provides methods for reducing SCAP protein levels in subjects with NAFLD. The present invention also provides methods for reducing the activity level of the hedgehog pathway in subjects with NAFLD.

[0138] In another aspect, the present invention provides a method for treating a subject with NAFLD. In one aspect, the present invention provides a method for treating a subject with a SCAP-related disease (e.g., fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, inflammation of the liver, hepatocyte necrosis, liver fibrosis, obesity, hepatocellular carcinoma, myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia, or non-alcoholic fatty liver disease (NAFLD)). The treatment method (and use) of the present invention comprises administering a therapeutically effective amount of an RNAi construct targeting a SCAP gene of the present invention, or a pharmaceutical composition comprising an RNAi construct targeting a SCAP gene of the present invention, or a vector comprising an RNAi construct targeting a SCAP gene of the present invention to a subject (e.g., a human).

[0139] In one aspect, the invention provides a method for preventing at least one symptom (e.g., presence of elevated signaling pathways, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider blood vessels, yellowing of the skin and eyes (jaundice), itching, fluid accumulation and swelling of the legs (edema), abdominal swelling (ascites), and confusion) in a subject with NAFLD. The method comprises administering a therapeutically effective amount of an RNAi construct (e.g., an RNAi construct), dsRNA, pharmaceutical composition, or vector of the invention to a subject, thereby preventing at least one symptom in a subject with a disorder that would benefit from decreased expression of a SCAP gene.

[0140] In another aspect, the present invention provides the use of a therapeutically effective amount of an RNAi construct of the present invention for treating a subject, such as a subject that benefits from reduction and / or inhibition of SCAP gene expression. In another aspect, the present invention provides the use of an RNAi construct (e.g., dsRNA) targeting a SCAP gene of the present invention, or a pharmaceutical composition comprising an RNAi construct targeting a SCAP gene in the preparation 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 suffering from a disorder that will benefit from reduction of SCAP gene expression (e.g., a SCAP-related disease)).

[0141] In another aspect, the invention provides use of the RNAi (eg, dsRNA) of the invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduction and / or inhibition of SCAP gene expression and / or SCAP protein production.

[0142] In another aspect, the invention provides use of an RNAi construct of the invention in the preparation of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduction and / or inhibition of SCAP gene expression and / or SCAP protein production, such as a SCAP-associated disease.

[0143] In one embodiment, a RNAi construct targeting SCAP is administered to a subject having a SCAP-related disease, such as non-alcoholic fatty liver disease (NAFLD), such that when the dsRNA agent is administered to the subject, the expression of the SCAP gene 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 %, 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 higher.

[0144] The methods and uses of the present invention include administering the compositions described herein such that the expression of the target SCAP gene is reduced within, for example, 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).

[0145] Administration of RNAi constructs according to the methods and uses of the present invention can result in a reduction in the severity, signs, symptoms and / or markers of a SCAP-related disease, such as non-alcoholic fatty liver disease (NAFLD), in patients with such disease or disorder. In this context, "reduction" refers to a statistically significant decrease in such levels. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. The efficacy of treating or preventing a disease can be assessed, for example, by measuring disease progression, disease remission, severity of symptoms, relief of pain, quality of life, drug dosage required to maintain a therapeutic effect, levels of disease markers, or any other measurable parameter suitable for a given disease being treated or targeted for prevention. It is well within the capabilities of those skilled in the art to monitor the efficacy of treatment or prevention by measuring any of such parameters or any combination of parameters. For example, the efficacy of NAFLD treatment can be assessed 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. It is entirely within the capabilities of those skilled in the art to monitor the efficacy of treatment or prevention by measuring any of such parameters or any combination of parameters. With respect to the administration of RNAi constructs targeting SCAP or their pharmaceutical compositions, "effective" against SCAP-related diseases indicates that administration in a clinically appropriate manner results in beneficial effects in at least a statistically significant portion of patients, such as improvement of symptoms, cure, alleviation of disease, extension of life, improvement of quality of life, or other effects generally considered to be positive by physicians familiar with the treatment of NAFLD and / or SCAP-related diseases and related causes.

[0146] The effect of treatment or prevention is evident when there is a statistically significant improvement in one or more disease state parameters, or when symptoms fail to worsen or develop where otherwise expected to occur. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease can be an indication of effective treatment. Experimental animal models for a given disease known in the art can also be used to judge the efficacy of a given RNAi drug or a formulation of the drug. In the case of using an experimental animal model, the efficacy of the treatment is demonstrated when a statistically significant reduction in a marker or symptom is observed.

[0147] Administration of the RNAi constructs can reduce the presence of SCAP protein levels, for example, in cells, tissues, blood, urine or other compartments of a patient 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%, %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or higher.

[0148] Before administering a full dose of RNAi construct, a smaller dose of the drug (e.g., 5% infusion) can be administered to the patient and adverse effects (e.g., allergic reactions) can be monitored. In another example, adverse immunostimulatory effects in the patient can be monitored, such as an increase in cytokine (e.g., TNF-α or IFN-α) levels.

[0149] Due to the inhibitory effect on SCAP expression, the composition according to the present invention or the pharmaceutical composition prepared therefrom can enhance the quality of life.

[0150] The RNAi constructs of the present invention can be administered in a "naked" form, where the modified or unmodified RNAi construct is directly suspended in an aqueous solvent or a suitable buffered solvent as "free RNAi". Free RNAi is administered in the absence of a pharmaceutical composition.

[0151] The RNAi can be in a pharmaceutical composition containing a suitable buffer solution. The buffer solution can include: acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmotic pressure of the buffer solution containing the RNAi construct can be adjusted so that the buffer solution is suitable for administration to a subject.

[0152] Alternatively, the RNAi constructs of the present invention can be administered in the form of a pharmaceutical composition, such as a liposomal formulation of the RNAi construct.

[0153] A subject that would benefit from reduction and / or inhibition of SCAP gene expression is a subject suffering from non-alcoholic fatty liver disease (NAFLD) and / or a SCAP-related disease or disorder as described herein.

[0154] Treatment of subjects who would benefit from reduction and / or inhibition of SCAP gene expression includes both therapeutic and prophylactic treatment.

[0155] The present invention also provides methods and uses of RNAi constructs or pharmaceutical compositions thereof for treating subjects (e.g., subjects suffering from a SCAP-related disease) who would benefit from reduction and / or inhibition of SCAP gene expression in conjunction with other drugs and / or other treatment methods, such as known drugs and / or treatment methods 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 useful for treating a SCAP-related disease, such as described elsewhere herein. For example, other therapeutic agents and treatment methods suitable for treating a subject who would benefit from reduced SCAP expression (e.g., a subject suffering from a SCAP-related disease) include: RNAi constructs targeting different portions of a SCAP gene, therapeutic agents, and / or procedures for treating a SCAP-related disease, or a combination of any of the foregoing.

[0157] In certain embodiments, a first RNAi construct targeting the SCAP gene is administered in combination with a second RNAi construct targeting a different portion of the SCAP gene. For example, the first RNAi construct comprises a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, 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 via a divalent or trivalent branched linker; and the second RNAi construct comprises a second sense strand and a second antisense strand forming a double-stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, 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 via a divalent or trivalent branched linker.

[0158] In one embodiment, all nucleotides of the first and second sense strands and / or all nucleotides of the first and second antisense strands comprise a modification.

[0159] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of: 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate esters, and nucleotides containing 5'-phosphate mimetics.

[0160] In certain 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 SCAP gene. The first RNAi construct targeting the SCAP gene and the second RNAi construct targeting a gene different from the SCAP gene (e.g., the SCAP gene) can be administered as part of the same pharmaceutical composition. Alternatively, the first RNAi construct targeting the SCAP gene and the second RNAi construct targeting a gene different from the SCAP gene (e.g., the SCAP gene) can be administered as part of different pharmaceutical compositions.

[0161] 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 using another method known in the art or described herein.

[0162] The present invention also provides a method for reducing and / or inhibiting the expression of SCAP in a cell using the RNAi construct of the present invention and / or a composition containing the RNAi construct of the present invention. In other aspects, the present invention provides an RNAi construct of the present invention and / or a composition containing the RNAi construct of the present invention for reducing and / or inhibiting the expression of the SCAP gene in a cell. In yet other aspects, the use of the RNAi construct of the present invention and / or a composition containing the RNAi construct of the present invention in the preparation of a drug for reducing and / or inhibiting the expression of the SCAP gene in a cell is provided. In still other aspects, the present invention provides an RNAi construct of the present invention and / or a composition containing the RNAi construct of the present invention for reducing and / or inhibiting the production of SCAP protein in a cell. In yet other aspects, the use of the RNAi construct of the present invention and / or a composition containing the RNAi construct of the present invention in the preparation of a drug for reducing and / or inhibiting the production of SCAP protein in a cell is provided. The method and use include contacting a cell with the RNAi construct of the present invention, and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the SCAP gene, thereby inhibiting the expression of the SCAP gene or inhibiting the production of the SCAP protein in the cell.

[0163] The reduction in gene expression can be assessed by any method known in the art. For example, the reduction in expression of SCAP can be determined by measuring the mRNA expression level of SCAP using methods routine to those of ordinary skill in the art (e.g., Northern blot, qRT-PCR), by measuring the protein level of SCAP using methods routine to those of ordinary skill in the art (e.g., Western blot, immunological techniques, flow cytometry, ELISA), and / or by measuring the biological activity of SCAP.

[0164] In the methods and uses of the invention, the cells may be contacted in vitro or in vivo, ie, the cells may be within a subject.

[0165] Cells suitable for treatment using the methods of the present invention may be any cells expressing the SCAP gene, such as cells from subjects with NAFLD or cells comprising an expression vector comprising the SCAP gene or a portion of the SCAP gene. Cells suitable for the methods and uses of the present invention may be mammalian cells, such as primate cells (such as human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (such as cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells), dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell.

[0166] 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%.

[0167] 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%.

[0168] The in vivo methods and uses of the present invention may include administering to a subject a composition containing an RNAi construct, wherein the RNAi construct includes a nucleotide sequence that is complementary to at least a portion of an RNA transcript of a SCAP gene of a mammal to be treated. 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 buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection. In one embodiment, the composition is administered by subcutaneous injection.

[0169] In some embodiments, administration is by depot injection. Depot injection can release RNAi in a consistent manner over an extended period of time. Therefore, depot injection can reduce the frequency of administration required to obtain a desired effect (e.g., a desired SCAP inhibition or treatment or prevention effect). Depot injection can also provide more consistent serum concentrations. Depot injection can include subcutaneous injection or intramuscular injection. In a preferred embodiment, depot injection is subcutaneous injection.

[0170] In some embodiments, administration is performed by a pump. The pump can 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. The infusion pump can be used for intravenous, subcutaneous, arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi construct to the subject.

[0171] 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 and site of administration can be selected to enhance targeting.

[0172] In one aspect, the present invention also provides a method for inhibiting the expression of a SCAP gene in a mammal (e.g., a human). The present invention also provides a composition comprising an RNAi construct targeting a SCAP gene in a mammalian cell, the RNAi construct being used to inhibit the expression of the SCAP gene in a mammal. In another aspect, the present invention provides the use of an RNAi construct targeting a SCAP gene in a mammalian cell in the preparation of a drug for inhibiting the expression of a SCAP gene in a mammal.

[0173] The method and use include administering a composition comprising an RNAi construct targeting the SCAP gene in mammalian cells to a mammal (eg, a human) and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of SCAP, thereby inhibiting the expression of the SCAP gene in the mammal.

[0174] The reduction in gene expression can be assessed in a peripheral blood sample of a subject to which RNAi is administered by any method known in the art (e.g., qRT-PCR as described herein). The reduction in protein production can be assessed by any method known in the art and by methods described herein (e.g., ELISA or Western blot). In one embodiment, a tissue sample serves as a tissue material for monitoring the reduction in SCAP gene and / or protein expression. In another embodiment, a blood sample serves as a tissue material for monitoring the reduction in SCAP gene and / or protein expression.

[0175] In one embodiment, in vivo verification of RISC-mediated target cleavage following administration of the RNAi construct is accomplished by performing 5'-RACE or modifications of protocols as known in the art (Lasham A et al. (2010) Nucleic Acid Res., 38(3) p-el9)(Zimmermann et al. (2006) Nature 441: 111-4).

[0176] It should be understood that all ribonucleic acid sequences disclosed herein can be converted into deoxyribonucleic acid sequences by replacing the uracil bases in the sequence with thymine bases. Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted into ribonucleic acid sequences by replacing the thymine bases in the sequence with uracil bases. Deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing a mixture of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein are included in the present invention.

[0177] In addition, any nucleic acid sequence disclosed herein can be modified with any combination of chemical modifications. It will be readily appreciated by those skilled in the art that, in some cases, such names as "RNA" or "DNA" for describing modified polynucleotides are arbitrary. For example, a polynucleotide comprising a nucleotide and a thymine base having a 2'-OH substituent on a ribose 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).

[0178] Therefore, the nucleic acid sequences provided herein (including but not limited to the nucleic acids in the sequence list) are intended to include nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to such nucleic acids with modified nucleic acid bases. By way of further example and without limitation, a polynucleotide having the sequence "ATCGATCG" includes any polynucleotide having such a sequence, whether modified or unmodified, including but not limited to such compounds comprising RNA bases, such as compounds having the sequence "AUCGAUCG" and compounds having some DNA bases and some RNA bases (such as "AUCGATCG"), and polynucleotides having other modified bases (such as "ATmeCGAUCG"), wherein meC represents a cytosine base including a methyl group at the 5 position.

[0179] The following examples, including experiments performed and results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

[0180] Incorporated by Reference

[0181] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, just as each individual publication, patent or patent application is specifically and individually incorporated herein by reference. However, the citation of references herein is not to be construed as an admission that such references are prior art to the present invention. To the extent that any definition or term provided in a reference incorporated by reference conflicts with the terms and discussion provided herein, the present terms and definitions shall prevail.

[0182] Equivalent application

[0183] The foregoing written description is considered sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples are described in detail in certain preferred embodiments of the invention and describe the best mode considered by the inventors. However, it will be appreciated that no matter how detailed the foregoing may appear in the text, the present invention may be practiced in many ways and should be interpreted in accordance with the appended claims and any equivalents thereof.

[0184] The following examples, including experiments performed and results achieved, are provided for illustrative purposes only and should not be construed as limiting the present invention.

[0185] Example 1: Selection, design and synthesis of modified SCAP siRNA molecules

[0186] Bioinformatics analysis of human SCAP transcripts (NM_012235) was used to confirm the identification and selection of optimal sequences for therapeutic siRNA molecules targeting sterol regulatory element binding protein (SREBP) cleavage activating protein (SCAP). Table 1 shows the sequences identified as having therapeutic properties. In various sequences, "invAb" is an inverted abasic nucleotide.

[0187] Table 1. siRNA sequences targeting SCAP

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] To improve the potency and in vivo stability of the SCAP siRNA sequence, chemical modifications were incorporated into the SCAP siRNA molecules. Specifically, 2'-O-methyl and 2'-fluoro modifications of the ribose were incorporated at specific positions within the SCAP siRNA. Phosphorothioate internucleotide bonds were also incorporated at the ends of the antisense and / or sense sequences. Table 2 below depicts the modifications in the sense and antisense sequences of each modified SCAP siRNA. Nucleotide sequences are listed in Table 2 according to the following notation: A, U, G, and C = corresponding ribonucleotides; dC and dG = corresponding deoxyribonucleotides; dT = deoxythymidine; a, u, g, and c = corresponding 2'-O-methyl ribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; [InvAb] is an inverted abasic residue; [Ab] is an abasic residue; GNA is a glycol nucleic acid, wherein the bases with the GNA backbone are shown as AgN, UgN, CgN, and GgN. The insertion of an "s" in the sequence indicates that two adjacent nucleotides are linked via a phosphorothioate diester group (e.g., a phosphorothioate internucleotide bond). Unless otherwise indicated, all other nucleotides are linked via a 3'-5' phosphodiester group. Each siRNA compound in Table 2 contains a duplex region of 19-21 base pairs with a 2 nucleotide overhang at the 3' end of both strands or a blunt end at one or both ends. Each [phosphate] has been linked to the following GalNAc structure:

[0194] ,

[0195] Where X=O or S.

[0196] Table 2. siRNA sequences with modifications targeting SCAP

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208] Example 2: Efficacy of selected SCAP siRNA molecules in RNA FISH assay

[0209] A panel of fully chemically modified siRNAs was prepared and tested in vitro for potency and selectivity in mRNA knockdown. Each siRNA duplex is composed of two strands, a sense strand or 'passenger' strand and an antisense strand or 'guide' strand, and is described in Example 1, wherein the native 2'-OH in the ribose of certain nucleotides is replaced. Optionally, the phosphodiester internucleotide bonds on one or both strands are replaced by phosphorothioates to reduce degradation by exonucleases.

[0210] RNA FISH (fluorescence in situ hybridization) assays were performed to measure SCAP mRNA knockdown with test siRNAs. Hep3B cells (purchased from ATCC) were cultured in minimum essential medium (MEM, Corning) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Corning). siRNA transfection was performed as follows: 1 μL of test siRNA and 4 μL of plain MEM were added to a PDL-coated CellCarrier-384Ultra assay plate (PerkinElmer) using BioMek FX (Beckman Coulter). Then, 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) pre-diluted in plain MEM (0.035 μL of RNAiMAX in 5 μL MEM) was dispensed into the assay plate using a Multidrop Combi reagent dispenser (ThermoFisher Scientific). After the siRNA / RNAiMAX mixture was incubated 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 plate was placed at room temperature for 20 minutes before being moved to the incubator. The cells were incubated at 37°C and 5% CO2 for 72 hours. ViewRNA ISH cell assays were performed using an in-house assembled automated FISH assay platform for liquid handling according to the manufacturer's protocol (ThermoFisher Scientific). Briefly, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 minutes at room temperature, permeabilized with detergent for 3 minutes at room temperature, and then treated with protease solution for 10 minutes at room temperature. Target-specific probe pairs (Thermo Fisher Scientific) were incubated for 3 hours, while preamplifiers, amplifiers, and labeled probes (Thermo Fisher Scientific) were incubated for 1 hour each. All hybridization steps were performed in a Cytomat 2C-LIN automated incubator (Thermo Fisher Scientific) at 40°C. After the hybridization reaction, cells were stained with Hoechst and CellMask blue (Thermo Fisher Scientific) for 30 minutes and then imaged on Opera Phenix (PerkinElmer). Images were analyzed using the Columbus Image Data Storage and Analysis System (PerkinElmer) to obtain the average spot count per cell. Spot counts were normalized using high (phosphate-buffered saline, Corning) and low (no target probe pair) control wells.Normalized values ​​were plotted against total siRNA concentration, and data were fit to a four-parameter sigmoidal curve model in the Genedata screener (Genedata) to obtain IC50 and maximal activity.

[0211] The results of RNA FISH assay of Hep3B cells are shown in Table 3. These values ​​indicate knockdown of SCAP mRNA.

[0212] Table 3. RNA FISH assay performed on Hep3B cells

[0213]

[0214]

[0215] Example 3: In vivo silencing studies to test the silencing efficacy of SCAP siRNA sequences

[0216] C57B16 male mice aged 9-10 weeks were purchased from Charles River Laboratories and housed according to the Amgen guidelines and the protocol of the Institutional Animal Care and Use Committee (IACUC). The animals were randomly divided into groups according to their body weight, and 6 animals were randomly assigned to each siRNA trigger sequence. On day 0, a single dose of 3 mg / kg body weight was administered subcutaneously to the group with PBS or with a specific siRNA compound. On day 29, mice were euthanized with CO2 and the left lobe of the liver was harvested from each animal. The tissue was cut into small pieces and immediately flash-frozen in liquid nitrogen for further downstream determination.

[0217] RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's guidelines. 2 ug of RNA was treated with DNase I (Promega) and quantitative PCR reactions were performed using Taqman RNA to CT One-Step Kit (Thermo Fischer Scientific). Gene-specific Taqman probes for mouse SCAP and GAPDH were used to quantify mRNA expression. GAPDH was used as an internal control. qPCR experiments were performed using the QuantStudio7 Flexible Real-Time PCR System from Invitrogen. Expression levels were calculated using the ΔCT method.

[0218] In vivo screening was performed on 9-10 week old Ob / Ob mice (Ob / Ob in a Bl6 background) from Jackson Laboratories using the same method as described for C57Bl6 mice.

[0219] All animal experiments described in this article were approved by the Institutional Animal Care and Use Committee (IACUC) of Amgen and were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition (National Research Council, USA). Committee on the Update of the Guide for the Care and Use of Laboratory Animals, National Institute of Laboratory Animal Research, and National Academy Press, USA (2011) Guide for the care and use of laboratory animals, 8th edition, National Academy Press, Washington, DC. Mice were housed individually in an air-conditioned room at 22°C ± 2°C with a 12-h light; 12-h dark cycle (0600-1800 hours). Unless otherwise stated, animals had free access to a regular chow diet (Envigo, 2920X) and water (purified by reverse osmosis) via an automated watering system. At the end of the experiment, blood was collected by cardiac puncture under deep anesthesia, followed by euthanasia by assisted physical means in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.

[0220] The relative knockdown data are shown in Table 4, which shows the relative knockdown at the 3 mg / kg dose on day 25. SCAP knockdown is the percentage reduction in SCAP mRNA levels.

[0221] Table 4. SCAP knockdown assay on day 25

[0222]

[0223]

[0224] Example 4: Efficacy of selected SCAP siRNA molecules in RNA FISH assay

[0225] A panel of fully chemically modified siRNAs was prepared and tested in vitro for potency and selectivity in mRNA knockdown. Each siRNA duplex is composed of two strands, a sense strand or 'passenger' strand and an antisense strand or 'guide' strand, and is described in Example 1, wherein the native 2'-OH in the ribose of certain nucleotides is replaced. Optionally, the phosphodiester internucleotide bonds on one or both strands are replaced by phosphorothioates to reduce degradation by exonucleases.

[0226] RNA FISH was performed as described in Example 2. Cells were incubated at 37°C and 5% CO2 for 72 hours. ViewRNA ISH cell assays were performed using an in-house assembled automated FISH assay platform for liquid handling, according to the manufacturer's protocol (Thermo Fisher Scientific). In brief, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 minutes at room temperature, permeabilized with detergent for 3 minutes at room temperature, and then treated with protease solution for 10 minutes at room temperature. Target-specific probe pairs (Thermo Fisher Scientific) were incubated for 3 hours, while preamplifiers, amplifiers, and labeled probes (Thermo Fisher Scientific) were incubated for 1 hour each. All hybridization steps were performed in a Cytomat 2C-LIN automated incubator (Thermo Fisher Scientific) at 40°C. After the hybridization reaction, cells were stained with Hoechst and CellMask blue (Thermo Fisher Scientific) for 30 minutes and then imaged on Opera Phenix (PerkinElmer). Images were analyzed using the Columbus Image Data Storage and Analysis System (PerkinElmer) to obtain the average spot counts per cell. Spot counts were normalized using high (phosphate-buffered saline, Corning) and low (no target probe pair) control wells. Normalized values ​​for total siRNA concentration were labeled and data were fitted to a four-parameter sigmoid curve model in the Genedata screener (Genedata) to obtain IC50 and maximum activity.

[0227] Results of RNA FISH assays of Hep3B cells are shown, Table 5 for duplexes D-2080 to D-2109, Table 6 for triggers D-2110 to D-2124, and Table 7 for triggers D-2125 to D2146. Negative values ​​of maximum activity indicate knockdown of activity.

[0228] Table 5. RNA FISH assay performed on Hep3B cells

[0229]

[0230]

[0231] Table 6. RNA FISH assay performed on Hep3B cells

[0232] Duplex number IC50(nM) Maximum activity D-2110 13.4 -74.1 D-2111 20.8 -73.8 D-2112 9.2 -71.4 D-2113 16.8 -70.2 D-2114 7.81 -80.3 D-2115 11.9 -62 D-2116 31.6 -58.5 D-2117 1.75 -79.6 D-2118 13.4 -66 D-2119 10.7 -65.5 D-2120 2.8 -56.4 D-2121 3.9 -51.7 D-2122 14.2 -67.9 D-2123 4.3 -63.3 D-2124 8.7 -62.2

[0233] Table 7. RNA FISH assay performed on Hep3B cells

[0234] Duplex number IC50(nM) Maximum activity D-2125 2.77 -63.4 D-2126 5.68 -74.4 D-2127 1.21 -81 D-2128 1.52 -69.1 D-2129 9.21 -90.2 D-2130 0.691 -84.6 D-2131 1.54 -85.4 D-2132 1.5 -83.4 D-2133 4.58 -89.3 D-2134 1.35 -70 D-2135 2.37 -75.4 D-2136 1.22 -71.5 D-2137 2.87 -67.4 D-2138 2.36 -62.4 D-2139 1.46 -68 D-2140 0.942 -71.7 D-2141 0.769 -79.7 D-2142 5.57 -60.9 D-2143 3.5 -64.5 D-2144 3.39 -64.1 D-2145 3.07 -75.3 D-2146 5.77 -72

[0235] Example 5: Screening of siRNA triggers modified with unstable bases

[0236] C57Bl6 / J male mice aged 9-10 weeks were obtained from Charles River Laboratories and adapted indoors. The mice were weighed and randomly divided into groups of 8 animals. The animals were subcutaneously administered with SCAP siRNA trigger at 3 mg per kg of body weight. Before administration, the reserve siRNA compound was diluted in a phosphate buffer without calcium and magnesium (Thermo Fisher Scientific, 14190-136). 30 days after siRNA treatment, the animals were euthanized and the liver was obtained. The newly isolated left lobe of the liver was immediately frozen in liquid nitrogen. According to the manufacturer's protocol, 30-50 mg of liver tissue was used to isolate RNA using the QIAcube HT instrument RNeasy96QIAcube HT kit. 2-4 ug RNA was treated with DNase (Promega, M6101) without RQ1 RNase. Real-time qPCR was performed on 10 ng of DNase-digested RNA using the TaqMan RNA to CT 1-step kit (Applied Biosystems) running on a Quant Studio real-time PCR machine. TaqMan probes for mouse SCAP (Mm01250176_m1) and GAPDH (4352932E) were used to calculate the fold change in SCAP expression in the SCAP siRNA-treated group compared to the PBS (buffer control) group. Data are presented as the percentage of knockdown in the siRNA-treated group relative to the PBS group. Five trigger sequences D-2040, D-2041, D-2042, D-2044, and D-2045 with various unstable modifications were tested. Unstable base modifications included GNA and abasic modification patterns. The data are shown in Table 8. In each case, the modification pattern containing unstable bases resulted in reduced SCAP mRNA expression compared to the parental trigger modification. The duplexes show % SCAP knockdown.

[0237] Table 8. Silencing in siRNAs with labile base modifications.

[0238]

[0239]

[0240] Example 6: Screening of siRNA triggers in C57Bl6 / J male mice

[0241] In addition, the D-2040, D-2045, D-2042, D-2041 and D-2044 sequences were modified using different chemical modification methods. These modified triggers were compared with the original trigger modification patterns. Groups 1 to 5 included changes in the trigger sequence D-2040 and the modification pattern. Groups 6 to 10 included changes in the trigger sequence D-2045 and the modification pattern. Groups 11 to 15 included changes in the trigger sequence D-2042 and the modification pattern. Groups 16 to 21 included changes in the trigger sequence D-2041 and the modification pattern. Groups 22 to 27 included changes in the trigger sequence D-2044 and the modification pattern. For the in vivo phase of the in vivo study, C57Bl6 / J male mice aged 13-15 weeks were obtained from Charles River Laboratories and adapted indoors. The mice were weighed and randomly divided into groups of 8 animals each. The animals were subcutaneously administered with SCAP siRNA trigger at 3 mg per kg of body weight. Before administration, the reserve siRNA compound was diluted in a phosphate buffer (Thermo Fisher Scientific, 14190-136) without calcium and magnesium. 30 days after siRNA treatment, the animals were euthanized and the liver was obtained. The newly isolated left lobe of the liver was immediately frozen in liquid nitrogen. According to the manufacturer's protocol, 30-50 mg of liver tissue was used to isolate RNA using QIAcube HT instrument RNeasy96QIAcube HT kit. 2-4 ug RNA was treated with DNase (Promega, M6101) without RQ1 RNase. Real-time qPCR was performed on 10 ng of DNase-digested RNA using TaqManRNA to CT 1-step kit (Applied Biosystems) running on Quant Studio real-time PCR machine. TaqMan probes for mouse SCAP (Mm01250176_m1) and GAPDH (4352932E) were used to calculate the fold change of SCAP expression in the siRNA-treated group compared to the PBS (buffer control) group. The data are shown in Table 9 and are expressed as the percentage of knockdown in the siRNA-treated group relative to the PBS group. As shown, different modification patterns produced different levels of silencing.

[0242] Table 9. Silencing in siRNAs with different modification patterns.

[0243]

[0244]

[0245] Example 7: siRNA-triggered silencing of SCAP in Ob / Ob animals

[0246] Male B6.V-Lep ob / J (632) mice, also known as Ob / Ob mice, 10-12 weeks old were obtained from Jackson Laboratory. After acclimation, the animals were randomly divided into groups of n=8. The mice were treated with SCAP siRNA triggers modified with chemical modifiers identified from the screening experiments in the above examples. The mice were subcutaneously administered 3 mg of siRNA per kg of body weight. The animals were sacrificed 20 and 30 days after siRNA administration. After euthanasia, the left lobe of the liver was isolated and immediately flash frozen in liquid nitrogen. 30-50 mg of liver tissue was used to isolate RNA using the QIAcube HT instrument RNeasy 96QIAcube HT kit according to the manufacturer's protocol. 2-4 ug RNA was treated with DNase without RQ1 RNase (Promega, M6101). Real-time qPCR was performed on 10 ng of DNase-digested RNA using the TaqMan RNA to CT 1-step kit (Applied Biosystems) running on a Quant Studio real-time PCR machine. TaqMan probes for mouse SCAP (Mm01250176_m1) and GAPDH (4352932E) were used to calculate the fold change in SCAP expression in the siRNA-treated groups compared to the PBS (buffer control) group. The data are shown in Table 10 and are expressed as the percentage of knockdown in the siRNA-treated groups relative to the PBS group.

[0247] Table 10. siRNA knockdown of SCAP in ob / ob animals

[0248] Duplex number Day 20 Harvest % Silence Day 30 Harvest % Silence D-2040 80.6 71.0 D-2126 84.4 77.2 D-2140 74.0 60.4 D-2145 80.7 74.5 D-2147 16.4 -21.3 D-2148 8.3 -1.0 D-2149 -5.0 -14.9 D-2150 22.4 -14.6

[0249] Example 8: Study on the efficacy of SCAP trigger

[0250] Prevention and restoration of NASH phenotype in efficacy models

[0251] [1] Amylin (AMLN) AMLYN model

[0252] The Amylin Liver NASH (AMLN) model was established by feeding 5-week-old obese male mice (Ob / Ob) obtained from Jackson Laboratories strain: B6.V-Lep ob / J (632) with a high-fat, high-cholesterol diet. The 45% fat, 36% carbohydrate and 2% cholesterol diet was obtained from Envigor, catalog number: TD170748. Regular water was replaced with a sugar solution containing 55% fructose and 46% glucose in water. Mice were randomly divided into 8 groups and treated with trigger D-2040 or trigger D-2147 or PBS. D-2147 is a seed sequence matched control for trigger D-2040, in which nucleotides 9 to 11 have been replaced. Mice were subcutaneously administered Q2D at 3 mg per kg body weight at weeks 8, 10 and 12 of the AMLN diet. Immediately prior to dosing, stock siRNA compounds were diluted in calcium- and magnesium-free phosphate buffer (Thermo Fisher Scientific, 14190-136). After 6 weeks of continuous SCAP silencing, mice were harvested at week 14 of the diet. During harvest, liver lobes were fixed in 10% neutral buffered formalin after euthanasia with isoflurane. Formalin-fixed lobes were further processed for hematoxylin and eosin (Dako, CS70030-2, CS70130-2), trichrome staining, and alpha smooth muscle actin (aSMA) expression using immunohistochemistry (IHC) according to the manufacturer's instructions. NASH readout was accomplished by scoring fibrosis and stellate cell activation and was performed by a board-certified pathologist.

[0253] The left lobe of liver is quick-frozen in liquid nitrogen. As described in detail in Example 7, quick-frozen tissue is further processed to be used for RNA extraction and the assessment of gene expression. In addition, liver triglyceride content is measured by homogenizing the quick-frozen liver tissue of 50-100mg in isopropanol. The sample is homogenized and incubated in ice for 1 hour, then rotated at 10000rpm for 10 minutes. The supernatant is transferred to a clean deep well 96 orifice plate. According to the manufacturer's instructions, triglyceride content is measured by colorimetric determination (Infinity triglyceride reagent, Thermo Fisher Scientific, TR22421) and using standard substance (Pointe Scientific, T7531-STD). Data are represented by milligrams of triglyceride per milligram of tissue.

[0254] Other endpoints captured during harvest included measuring liver weight. The ratio of whole liver weight (g) to terminal body weight (g) was analyzed to monitor changes in liver mass. SCAP silencing inhibited PCSK9 expression. Serum PCSK9 levels were measured as a biomarker using an ELISA assay (R&D Systems, MPC900).

[0255] Figure 1 A depicts the expression of SCAP mRNA expressed as fold change relative to the PBS control group. The trigger D-2040 treatment group achieved approximately 85% SCAP silencing (85.3%), while there was no significant change in the D-2147 treatment group. Figure 1 B shows a significant decrease in terminal liver weight:body weight ratio in trigger D-2040 treated mice. Figure 1 C shows that liver triglycerides decreased in mice treated with trigger D-2040, but remained unchanged in the D-2147 treated group. Figure 1 In D, serum PCSK9 levels were measured. High-efficiency SCAP silencing significantly reduced serum PCSK9 levels. Figure 1 E and Figure 1 Pathological readouts of fibrosis (trichrome staining) and stellate cell activation (aSMA immunohistochemistry) are shown in F. SCAP silencing using trigger D-2040 significantly reduced fibrosis scores, indicating improvement in NASH outcomes. Statistical significance was measured by one-way ANOVA with Dunnett's multiple comparison test, where asterisks indicate adjusted p-values ​​(****p-value < 0.0001.***p-value < 0.001).

[0256] [2] ALIOS model

[0257] The American Lifestyle-Induced Obesity Syndrome mouse model (ALIOS) was also used to test the efficacy of SCAP triggers. Five-week-old C57B16 male mice obtained from Charles River Laboratories were fed the ALIOS diet, which is similar to the AMLN diet except for reduced cholesterol content. The ALIOS diet contains 0.2% cholesterol and is obtained from Envigol (Catalog No. TD130885). Regular water was replaced with a sugar solution containing 55% fructose and 46% glucose in water. After 18 weeks of feeding the animals on the ALIOS diet, mice were subcutaneously administered 3 mg per kg of body weight every two weeks for 6 weeks. Mice were randomized into groups of n=4-5 and treated with trigger D-2040, trigger D-2042, or PBS. Mice were harvested after 6 months of diet and 6 weeks of SCAP silencing. Similar to previous efficacy studies, endpoint analyses included: SCAP messenger levels, terminal liver weight / body weight ratio, liver triglyceride levels, serum PCSK9 levels and pathological readout of fibrosis using trichrome staining, and stellate cell activation using immunohistochemical staining for alpha smooth muscle actin.

[0258] Figure 2 A shows SCAP mRNA expression. Data are presented as fold change relative to the PBS group. Both SCAP triggers D-2040 and D-2042 exhibited more than 85% reduction in SCAP mRNA. Figure 2 In B, a significant decrease in the terminal liver weight / body weight (LW / BW) ratio was observed in the groups treated with SCAP triggers D-2040 and D-2042. Figure 2 C shows the liver triglyceride levels in different groups. Administration of SCAP triggers D-2040 and D-2042 significantly reduced liver triglyceride content compared to the buffer control group. Figure 2 Serum PCSK9 was measured using the ELISA kit described above in D. The serum PCSK9 level in the SCAP siRNA-treated group was significantly decreased compared to the PBS group. Figure 2 E and Figure 2 F shows the readout of fibrosis pathology measured by trichrome staining and alpha smooth muscle actin immunostaining indicating stellate cell activation. Either readout showed a decrease after SCAP silencing, indicating restoration of the NASH phenotype after SCAP silencing. Statistical significance was measured by one-way ANOVA with Dunnett's multiple comparison test, where asterisks indicate adjusted p-values ​​(****p<0.0001, ***p<0.005, **p<0.01 and *p<0.05)

[0259] Example 9: Testing the efficacy of SCAP siRNA in the treatment of hepatocellular carcinoma using the DIAMOND model

[0260] More than 50% of patients with hepatocellular carcinoma suffer from non-alcoholic fatty liver disease. To test the efficacy of SCAP siRNA in preventing further progression of HCC, we implemented a model in which a long-term NASH diet exhibits HCC without the use of chemical modifiers. Such models better represent human pathophysiology. One such model is the diet-induced animal model of non-alcoholic fatty liver disease (or DIAMOND). It was established using a unique syngeneic animal strain obtained from the C57Bl / 6J and 1291SvImJ backgrounds. Male mice from this intergenic population were fed a high-fat, high-carbohydrate diet (42% kcal from fat) containing 0.1% cholesterol from the 8th week of age. In addition, drinking water was also replaced by a high fructose-glucose solution. After 32 weeks on this diet, the model developed HCC. By the 51st week, DIAMOND model liver tissue exhibited large areas of tumors and altered lesions within hepatocytes. The model is also highly permeable. To test efficacy, SCAP siRNA and vehicle controls were administered at the 40th week of the diet. Mice were re-administered with vehicle or SCAP siRNA at regular intervals for 6-10 weeks to ensure reduction of SCAP gene expression. Endpoint analyses included pathological examination of the metastatic index of hepatocellular tumor burden, assessment of tumor proliferation using Ki67 expression, and the extent of tumor angiogenesis obtained using CD31 expression. In addition, qPCR and protein analysis were evaluated to confirm efficient silencing of targets and downstream pathways.

[0261] Example 10: Evaluation of SCAP siRNA in the Huh-7 liver xenograft model of HCC

[0262] SCAP siRNA was evaluated using an orthotopic Huh-7 liver xenograft model. Six-week-old BALB / c athymic nude mice were injected intrahepatically with 1 million Huh-7 cells suspended in cell culture medium containing 33% Matrigel. Mice were then divided into groups for treatment with vehicle or SCAP siRNA. Vehicle or SCAP siRNA was re-administered at regular intervals (e.g., every two weeks) to ensure continued reduction of SCAP mRNA. At each time point after initial vehicle or siRNA treatment (e.g., week 4), mice were euthanized and livers were harvested and fixed in 4% paraformaldehyde. Tumor burden was measured to understand the efficacy of SCAP siRNA treatment. In addition, qPCR and protein analysis were evaluated to confirm efficient silencing of the target.

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 sequence listed in Table 1 or Table 2, and wherein the RNAi construct inhibits the expression of SREBP cleavage activating protein (SCAP).

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

3. The RNAi construct of any one of the preceding claims, wherein the sense strand comprises a region of at least 15 consecutive nucleotides having no more than 3 nucleotide differences from the antisense sequence listed in Table 1 or Table 2.

4. A pharmaceutical composition comprising the RNAi construct according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier, excipient, or diluent.

5. Use of the RNAi construct of any one of claims 1 to 3 in the preparation of a medicament for reducing SCAP expression in a patient in need thereof, comprising administering the RNAi construct of any one of claims 1 to 3 to the patient.

6. The use of claim 5, wherein the expression level of SCAP in hepatocytes in the patient after administration of the RNAi construct is reduced compared to the expression level of SCAP in the patient not receiving the RNAi construct.

7. Use of the RNAi construct of any one of claims 1 to 3 or the pharmaceutical composition of claim 4 in the preparation of a medicament for treating a subject suffering from a SCAP-related disease, comprising administering the RNAi construct of any one of claims 1 to 3 or the pharmaceutical composition of claim 4 to the subject.

8. The use of claim 7, wherein administration of the RNAi construct of any one of claims 1 to 3 or the pharmaceutical composition of claim 4 to the subject results in a decrease in the expression of the SCAP gene by at least about 10%.

9. The method of claim 7, wherein the disease is selected from the group consisting of fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocellular necrosis, hepatocellular carcinoma, liver fibrosis, obesity, myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia, or non-alcoholic fatty liver disease (NAFLD).

10. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of at least 15 consecutive nucleotides that differs by no more than 3 nucleotides from the antisense sequence listed in Table 1 or Table 2, the RNAi construct being for use in a method for treating a SCAP-related disease, the method comprising administering to the subject the RNAi construct of any one of claims 1 to 3 or the pharmaceutical composition of claim 4.

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