RNAi agent for inhibiting SCAP gene expression and preparation method and application thereof

Abstract

The present invention relates to an RNAi agent for inhibiting SCAP gene expression, a preparation method and use thereof, the RNAi agent being capable of inhibiting SCAP gene expression to treat SCAP gene related diseases and disorders, such as treatment of elevated blood lipid levels.

Description

Technical Field

[0001] The present invention relates to the field of medicine. More specifically, the present invention relates to an RNAi agent for inhibiting the expression of the SCAP gene, its preparation method and uses, such as treating elevated blood lipid levels. Background Art

[0002] Sterol regulatory element-binding protein cleavage-activating protein (SCAP) is a membrane protein located on the endoplasmic reticulum. Its main function is to transport SREBP-1c to the Golgi apparatus for hydrolysis and activation, releasing the active fragment into the nucleus to play a role.

[0003] RNAi agents targeting the function of SCAP can reduce the level of SCAP and treat elevated blood lipid levels, such as: WO2008036638A2, WO2017100542A1, WO2023201043A1, and CN113825839A, CN113727732A, CN113924368A. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides an RNAi agent for inhibiting the expression of the SCAP gene, its preparation method and uses. The RNAi agent has a good inhibitory effect on the expression of the SCAP gene.

[0005] In a first aspect, the present invention provides an RNAi agent for inhibiting the expression of the SCAP gene, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially complementary, and the sense strand and the antisense strand are selected from the sequences shown in Table 1.

[0006] Wherein, the sense strand and the antisense strand may be partially, substantially or completely complementary to each other. For example, the sense strand and the antisense strand may be 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary.

[0007] Wherein, the lengths of the nucleotides of the sense strand and the antisense strand may be the same or different. For example, the sense strand comprises 19 nucleotides, the antisense strand comprises 21 nucleotides, or the sense strand comprises 19 nucleotides and the antisense strand also comprises 19 nucleotides, or the sense strand comprises 21 nucleotides and the antisense strand comprises 19 nucleotides, or the sense strand comprises 21 nucleotides and the antisense strand comprises 21 nucleotides.

[0008] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand comprise at least one modified nucleotide.

[0009] Wherein, the modified nucleotides are selected from:

[0010] alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxynucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphonate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinyl phosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, dithiophosphonate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), glycol nucleic acids (GNA); wherein, the alkyl nucleotides are selected from methyl nucleotides and ethyl nucleotides; the glycol nucleic acids include (S)-glycol nucleic acid ((S)-GNA) and (R)-glycol nucleic acid ((R)-GNA).

[0011] Wherein, both the sense strand and the antisense strand of the RNAi agent include at least one modified nucleotide. In a specific embodiment, the sense strand includes at least one modified nucleotide, and the nucleotides of the antisense strand are not modified, or the nucleotides of the sense strand are not modified, and the antisense strand includes at least one modified nucleotide. In a specific embodiment, each nucleotide of the sense strand is modified, and each nucleotide of the antisense strand is also modified.

[0012] As a preferred technical solution of the present invention, the sense strand and the antisense strand have the modified forms shown in the following formula:

[0013] Sense strand: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm;

[0014] Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0015] Wherein: "N" represents a nucleotide; m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide; s indicates that there is a phosphorothioate linkage between the two nucleotides adjacent to the letter s on the left and right.

[0016] As a preferred technical solution of the present invention, the modified RNAi agent is selected from the sequences shown in Table 2.

[0017] As a preferred technical solution of the present invention, the RNAi agent further includes a targeting ligand, and the targeting ligand is connected to the sense strand and / or the antisense strand.

[0018] As a preferred technical solution of the present invention, the targeting ligand comprises an N-acetyl-galactosamine (GalNAc) moiety.

[0019] As a preferred technical solution of the present invention, the targeting ligand is selected from:

[0020]

[0021] wherein, represents the siRNA ligation site, R 2 represents siRNA, and Nu represents siRNA.

[0022] As a preferred technical solution of the present invention, the targeting ligand is linked to the 3' or 5' end of the sense strand.

[0023] As a preferred technical solution of the present invention, the targeting ligand is linked to the 3' or 5' end of the antisense strand.

[0024] In some specific embodiments, the targeting ligand is linked to the 5' end of the sense strand. In some specific embodiments, the targeting ligand is linked to the 3' end of the sense strand. In some specific embodiments, the targeting ligand may also be internally linked to nucleotides on the sense strand and / or antisense strand of the RNAi agent. In some specific embodiments, the targeting ligand may also be linked to the double-stranded RNAi agent via a linker. For example, the targeting ligand may be linked to the 3' or 5' end of the sense strand via a linker, or the targeting ligand may be linked to the 3' or 5' end of the antisense strand via a linker, or the targeting ligand may be internally linked to nucleotides on the sense strand and / or antisense strand of the double-stranded RNAi agent via a linker.

[0025] In a second aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of the SCAP receptor, and the pharmaceutical composition comprises the RNAi agent as described above.

[0026] In a third aspect, the present invention further provides the use of the RNAi agent as described above in the preparation for treating SCAP receptor-related diseases, disorders or symptoms.

[0027] As a preferred technical solution of the present invention, the diseases are selected from treating elevated blood lipid levels, etc.

[0028] The RNAi agent provided by the present invention for inhibiting the expression of the SCAP gene has good inhibitory activity against the SCAP gene and can be used for preventing and / or treating related diseases mediated by the SCAP gene. Detailed implementation manners

[0029] The present invention will be further described in detail below with reference to the embodiments, but the implementation manners of the invention are not limited thereto.

[0030] As used herein, the term "comprising" is intended to mean the phrase "comprising (but not limited to)" and may be used interchangeably with that phrase, unless the context clearly indicates otherwise.

[0031] The term "or" as used herein is intended to mean the term "and / or" and may be used interchangeably with that term, unless the context clearly indicates otherwise.

[0032] As used herein, the terms "sequence" and "nucleotide sequence" mean the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0033] In the present invention, the term "RNAi agent" refers to a complex of ribonucleic acid molecules having a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands, which have "sense" and "antisense" orientations relative to the target RNA.

[0034] In the present invention, "complementary" has the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand.

[0035] The term "antisense strand" generally refers to the strand of an RNAi agent that includes a region substantially complementary to the target sequence. As used herein, the term "complementary region" generally refers to the region on the antisense strand that is substantially complementary to the sequence defined in the present application (e.g., the target sequence). When the complementary region is not completely complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides at the 5' end and / or 3' end.

[0036] The term "sense strand" generally refers to the strand of an RNAi agent that includes a region substantially complementary to the region defined as the antisense strand herein. The "sense" strand is sometimes referred to as the "sense" strand, the "passenger" strand, or the "anti-guide" strand. By virtue of their sequences, the antisense strand targets the desired mRNA, while the sense strand targets a different target. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. The incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications on the sense strand or using a 5' end cap.

[0037] In the present invention, the modified nucleotides include, but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinyl phosphonate deoxyribonucleotides (VP), phosphorothioate nucleotides, dithiophosphonate nucleotides, locked nucleic acids (LNA), morpholino oligonucleotides (PMO), inverted abasic deoxyribose residues (invAb), glycol nucleic acids (GNA).

[0038] Among them, the alkyl-modified nucleotides, such as 2'-methyl nucleotides, have the structure: 2'-ethyl nucleotides have the structure: 2'-methoxy-modified nucleotides have the structure: 2'-methoxyethyl nucleotides have the structure: 2'-O-methoxyethyl nucleotides have the structure:

[0040] 2'-fluoro nucleotides have the structure: Vinyl phosphonate nucleotides (VP) have the structure: Phosphorothioate nucleotides (S) have the structure: Phosphate nucleotides (p) have the structure: 2'-deoxyribonucleotides have the structure: Inverted abasic deoxyribose residues (invAb) have the structure:

[0041] Glycol nucleic acids (GNA), including (S)-glycol nucleic acids ((S)-GNA), have the structure: And (R)-glycol nucleic acids ((R)-GNA) have the structure: The structure of TNA is:

[0042] 3'-OMe-2',5'-PS has the structure

[0043] Among them, Base represents a base, R represents an alkyl group, Me represents a methyl group, and Et represents an ethyl group.

[0044] The term "locked nucleic acid" refers to a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2'-carbon and the 4'-carbon. This structure effectively "locks" the ribose in the 3'-endo conformational state. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1): 439-447; Mook, O.R. et al., (2007) Mol Cancer Ther 6(3): 833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12): 3185-3193).

[0045] Representative U.S. patents for preparing locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are incorporated herein by reference.

[0046] The structure of locked nucleic acid is as follows:

[0047]

[0048] In certain embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides containing morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0049] The term "morpholino" refers to a sugar substitute having the following formula:

[0050]

[0051] In certain embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the above morpholino structure. Such sugar substitutes are referred to herein as "modified morpholino groups".

[0052] In the present invention, unless otherwise specified, the capital letters C, G, U, and A represent the base composition of nucleotides. The lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a nucleotide modified with a methoxy group; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a nucleotide modified with a fluoro group; LNA indicates that the nucleotide adjacent to the right of it is a nucleotide modified with locked nucleic acid (LNA); GNA indicates that the nucleotide adjacent to the left of it is a nucleotide modified with GNA; the lowercase letter s indicates that there is a phosphorothioate linkage between the two nucleotides on the left and right of the letter; VP indicates that the nucleotide adjacent to the right of the letter VP is a nucleotide modified with vinyl phosphate. invAb represents an inverted abasic deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents deoxyadenosine nucleotide; dT represents deoxythymidine nucleotide; dU represents deoxyuridine nucleotide; dC represents deoxycytidine nucleotide; dG represents deoxyguanosine nucleotide.

[0053] It should be emphasized that the "modification" of the nucleotides described in the present disclosure includes, but is not limited to, the above examples, and the nucleotides can also be replaced with other nucleotides, for example, (S)-glycerol nucleic acid, etc.

[0054] The term "targeting ligand" can include naturally occurring substances, such as proteins (e.g., human serum albumin (HAS), low density lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, mimetic peptide polyamine, dendrimeric polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or α-helical peptide.

[0055] Targeting ligands can also be cell or tissue targeting agents that bind to a specified cell type, such as renal cells, e.g., lectins, glycoproteins, lipids or proteins, e.g., antibodies. Targeting groups can be thyroid stimulating hormone, melanocyte stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimetics.

[0056] Targeting ligands can also be proteins, e.g., glycoproteins, or peptides, e.g., molecules having specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to a specified cell type, such as hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, saccharides, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. Ligands can be, e.g., lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-KKB.

[0057] Targeting ligands can be substances, such as drugs, that can increase the uptake of iRNA agents into cells, e.g., by disrupting the cytoskeleton of the cell (e.g., by disrupting cell microtubules, microfilaments and / or intermediate filaments). Drugs can be, e.g., taxol, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, spongistatin A, phalloidin, swinholide A, indanocine or myoservin.

[0058] The pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The formulations can conveniently be in unit dosage form and can be prepared by any methods well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier substance to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally, on a percentage basis, the amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0059] The term "prevention and / or treatment" includes not only the prevention and / or treatment of diseases, but also generally includes preventing the onset of diseases, slowing or reversing the progression of diseases, preventing the onset of one or more symptoms associated with diseases, reducing and / or alleviating one or more symptoms associated with diseases, reducing the severity and / or duration of diseases and / or any symptoms associated therewith and / or preventing further increase in the severity of diseases and / or any symptoms associated therewith, preventing, reducing or reversing any physical damage caused by diseases, and any pharmacological effects generally beneficial to the patients being treated. For the nucleic acids or pharmaceutical compositions of the present application to form viable therapeutic agents, it is not necessary to achieve complete cure or eradication of any symptoms or manifestations of diseases. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but it is not necessary to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, for a treatment administered prophylactically to constitute a viable prophylactic agent, it is not necessary to be completely effective in preventing the onset of the condition. Simply reducing the impact of the disease in a subject (e.g., by reducing the number or severity of its symptoms, or by enhancing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood of the occurrence or worsening of the disease is sufficient.

[0060] The terms "disease" or "condition" are used interchangeably and generally refer to any deviation of a subject from the normal state, such as any change in the state of the body or certain organs, which impedes or disrupts the performance of functions, and / or causes symptoms such as discomfort, dysfunction, pain or even death in the diseased or exposed person. A disease or condition may also be referred to as distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, indisposition or affectation.

[0061] The term "inhibit" can be used interchangeably with "reduce", "silence", "down-regulate", "stop" and other similar terms and includes inhibition at any level.

[0062] The term "SCAP" refers to sterol regulatory element-binding protein cleavage-activating protein (SREBP cleavage-activating protein, SCAP), which is a membrane protein located on the endoplasmic reticulum. Its main function is to be responsible for transporting SREBP-1c to the Golgi apparatus for hydrolysis and activation, releasing the active fragment into the nucleus to play a role. Among them, the term "SCAP-related diseases, conditions" refers to liver disease-related diseases, such as treating elevated blood lipid levels.

[0063] The term "SCAP" includes human SCAP, the amino acid and nucleotide sequences of which can be found, for example, in GenBank accession number NM_012235.4; mouse SCAP, the amino acid and nucleotide sequences of which can be found, for example, in GenBank accession number NM_001001144.3; rat SCAP, the amino acid and nucleotide sequences of which can be found, for example, in GenBank accession number: NM_001100966.2; cynomolgus monkey SCAP, the amino acid and nucleotide sequences of which can be found, for example, in GenBank accession number: XM_045384941.1.

[0064] In the present invention, the phrase "inhibiting the expression of the SCAP gene" includes inhibiting any SCAP gene (e.g., mouse SCAP gene, rat SCAP gene, monkey SCAP gene, or human SCAP gene, together with the expression of variants or mutants encoding the SCAP gene).

[0065] "Inhibiting the expression of the SCAP gene" includes inhibition of the SCAP gene at any level, e.g., at least partially arresting the expression of the SCAP gene, such as inhibiting 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%.

[0066] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents found in the present invention with pharmaceutically acceptable acids or bases.

[0067] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and is non-toxic and side-effect-free to the host or patient. Representative carriers include water, oils, vegetables and minerals, paste bases, lotion bases, ointment bases, etc. These bases include suspending agents, thickening agents, transdermal promoters, etc. Their formulations are well known to those skilled in the art of the cosmetic field or the topical drug field. For other information on carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the content of which is incorporated herein by reference.

[0068] The term "pharmaceutically acceptable excipient" refers to substances intentionally included in a drug delivery system in addition to the active pharmaceutical ingredient (API, therapeutic product, such as the SCAP gene). Excipients do not exert or are not intended to exert a therapeutic effect at the intended dose. Excipients can serve the following functions: a) assist in the handling of the drug delivery system during preparation, b) protect, support or enhance the stability, bioavailability or patient acceptability of the API; c) assist in product identification; and / or d) enhance any other properties of the API in terms of overall safety, effectiveness or delivery during storage or use.

[0069] Among them, excipients include (but are not limited to): absorption enhancers, anti-adhesives, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextrans, dextroses, diluents, disintegrants, emulsifiers, swelling agents, fillers, flavoring agents, glidants, wetting agents, oils, polymers, preservatives, saline solutions, salts, solvents, saccharides, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium dodecyl sulfate and magnesium stearate, flavor correctors and fragrances.

[0070] The terms "active ingredient", "therapeutic agent", "active substance" or "active agent" refer to a chemical entity that can effectively treat a target disorder, disease or condition.

[0071] Example 1 Synthesis of Targeting Ligands

[0072]

[0073] Among them, the synthesis route of the targeting ligand L96 refers to the literature with the international patent publication number WO2009073809. The targeting ligand L96 can be linked to siRNA through a phosphate group, a phosphorothioate group or another linking group, indicating the siRNA linkage site.

[0074]

[0075] Among them, the synthesis route of NAG37 can refer to the literature with the patent number CN201780042047.6, where indicating the siRNA linkage site.

[0076]

[0077] M10 (Conjugates 9 and 10)

[0078] Among them, the synthesis route of M10 (Conjugates 9 and 10) can refer to the literature with the patent number CN202210705962.1, R 2Represents siRNA.

[0079]

[0080] Among them, the synthesis route of L10 can refer to the synthesis route of the patent with the patent publication number WO2019105414A1, where Nu represents siRNA.

[0081]

[0082] Among them, the synthesis route of GLS-15 can refer to the synthesis route of the patent with the patent publication number CN202280016262.X, where Represents the siRNA ligation site.

[0083]

[0084] Among them, the synthesis route of GL6 can refer to the synthesis method of compound 6 in the patent document WO2023241591, Represents the siRNA ligation site.

[0085] Example 2 Synthesis of RNAi Agent

[0086] An RNAi agent was prepared using an OligoMakerApS 192RNA synthesizer (produced in Denmark). Among them, the specific synthesis route can refer to the patent document CN202080041291.2, and the RNAi agent sequence is shown in Table 1.

[0087] Table 1 is the sequence list of the RNAi agent

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Synthesis of the Modified RNAi Agent in Example 3

[0110] A modified RNAi agent was prepared using an OligoMaker ApS 192 RNA synthesizer (manufactured in Denmark). Specifically, the double-stranded siRNA analog shown in Table 1 was prepared into a double-stranded siRNA analog with the modified form shown in the following formula:

[0111] Sense strand: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNm;

[0112] Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0113] Wherein: "N" represents a nucleotide, such as A, G, U, and C;

[0114] m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-O-methyl modified A, U, G, and C, respectively; f indicates that the nucleotide adjacent to its left is a 2'-fluoro modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-fluoro modified A, U, G, and C, respectively;

[0115] s indicates that there is a phosphorothioate linkage between the two nucleotides adjacent to the letter s on the left and right.

[0116] For the specific synthesis route, reference can be made to the patent document CN202080041291.2, and the sequences of the modified RNAi agents are shown in Table 2.

[0117] Table 2 is the sequence list of the modified RNAi agents

[0118]

[0119]

[0120]

[0121]

[0122] Among them, A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Tm = 2'-O-methylthymidine-3'-phosphate; Ams = 2'-O-methyladenosine-3'-thiophosphate; Ums = 2'-O-methyluridine-3'-thiophosphate; Cms = 2'-O-methylcytidine-3'-thiophosphate; Gms = 2'-O-methylguanosine-3'-thiophosphate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Afs = 2'-fluoroadenosine-3'-thiophosphate; Ufs = 2'-fluorouridine-3'-thiophosphate; Cfs = 2'-fluorocytidine-3'-thiophosphate; Gfs = 2'-fluoroguanosine-3'-thiophosphate; m = 2'-O-methyl; f = 2'-fluoro; s = thiophosphate linkage.

[0123] Synthesis of the RNAi agent compound in Example 4

[0124] An OligoMaker ApS192 RNA synthesizer (produced in Denmark) was used to prepare the modified RNAi agent. Then, the targeting ligand L96 in Example 1 was linked to the 3'-end of the sense strand of the modified RNAi agent in Table 2. For the specific synthesis route, reference can be made to the patent document CN202080041291.2, and the content of this patent document can be incorporated into this application by reference.

[0125]

[0126] Example 5 In vitro activity test of SCAP siRNA in Huh7 cells

[0127] The effect of siRNA on the expression of SCAP gene in HuH7 cells was detected by qPCR experiment (Taqman probe method).

[0128] HuH7 cells were cultured in DMEM (Pricella - PM150210) medium containing 10% fetal bovine serum (Corning, 35 - 081 - CV). HuH7 cells in the logarithmic growth phase were taken and seeded into 96 - well plates at a density of 1.0×10 4 / well, and incubated overnight in an incubator at 37°C and 5% CO2. The medium in the wells was replaced with 40 μL of Opti - mem (Gibco - 31985 - 062). siRNA was serially diluted to 10 - fold of the final working concentrations (1 nM and 0.02 nM) with Opti - mem, and then 15 μL / well was added to the dilution plate. 54 μL of Lipofectamine 2000 (Invitrogen, 52887) and 846 μL of Opti - mem were incubated at room temperature for 5 min, and then 15 μL / well was added to the siRNA wells, mixed well, and incubated at room temperature for 18 min. 10 μL / well of the liposome - siRNA complex was added to the corresponding cell wells and cultured in the incubator. After 6 h of transfection, the complete medium was replaced, and after incubating in the incubator for 48 h, total cellular RNA was extracted using the Baypure magnetic bead method tissue cell total RNA extraction kit (BayBio, TIRM - 48 - K - D). Then, the extracted cellular RNA was reverse - transcribed into cDNA using TransScript Uni All - in - One First - Strand cDNA Synthesis SuperMix for qPCR (Trans - AU341 - 02 - V2). Finally, qPCR experiment was performed on the obtained cDNA using EZ - Probe qPCR Master Mix - UDG (EZB - EZB - Probe - U2).

[0129] Example 6 detected the effect of siRNA on the expression of SCAP gene in HuH7 cells by q - PCR experiment (Taqman probe method).

[0130] To determine the inhibition efficiency at the mRNA level, after transfection of SCAP siRNA into HuH7 cells, q-PCR experiments were performed to measure the expression level of SCAP mRNA. Specifically, HuH7 cells were cultured in DMEM medium (Gibco, 11965-092) containing 10% fetal bovine serum (ExCell Bio, FSP500), 1% glutamine (Gibco, 35050061), 1% NEAA (Gibco, 11140050), and 1% penicillin-streptomycin (HyClone, SV30010). HuH7 cells in the logarithmic growth phase were taken and seeded in a 96-well cell culture plate at a density of 1×10 4 cells / well. At the same time of seeding, siRNA was mixed with Lipofectamine TM RNAiMAX (INVITROGEN, 13778150) so that the final concentration of the RNAi agent mixture was 1 nM and 0.02 nM, and then the RANi agent mixture was transfected into the cells. The cells were cultured overnight in a 37°C, 5% CO2 incubator, and 2 replicate wells were measured in parallel. At the same time, a cell control group without compounds containing Lipofectamine TM RNAiMAX was set up. After 48 h of transfection, the medium was removed, and total cellular RNA was extracted (QIAGEN-74182) and reverse transcribed (Vazyme-R323-1). The target cDNA was detected by the TaqMan method, and GAPDH cDNA was detected simultaneously as an internal control for parallel detection.

[0131] The expression level of the target gene mRNA in each sample was calculated by the ΔΔCT relative quantification method. The relative expression level of the target gene was expressed using 2 -ΔΔCt . Specifically, the CT value of the target gene in each sample was subtracted from the CT value of the internal reference gene (GAPDH) to obtain the ΔCT value. Subsequently, the ΔCT of the dosing group (sample) was subtracted from the ΔCT value of the control group containing only the transfection reagent (RNAiMAX Control) to obtain ΔΔCT. Finally, ΔΔCT was subjected to 2 -ΔΔCt conversion to finally obtain the relative expression level of MARC1 mRNA (value of sample). The inhibition rate of the MARC1 gene mediated by the siRNA analog was calculated according to the following formula. SCAP inhibition rate % = (1 - value of sample / Average value of RNAiMAX Control) * 100.

[0132] The test results are shown in Table 3 below,

[0133] Table 3

[0134] Number Inhibition Rate % - 1nm Number Inhibition Rate % - 1nm S1M A S62M A S3M A S63M A S4M A S67M A S5M A S78M A S6M A S82M A S7M A S89M A S8M A S95M A S9M A S96M A S19M A S98M A S21M A S101M A S22M A S113M A S30M A S114M A S38M A S116M A S40M A S117M A S49M A S119M A S52M A S129M A S53M A S130M A S55M A S131M A S61M A

[0135] Among them, inhibition rate %: A > 70%.

[0136] Example 7

[0137] In vitro test of SCAP RNAi agent in Huh7 cells: The exon region (including 5'UTR and 3'UTR sequences) of the human SCAP (NM_012235) gene was cloned into the reporter-based screening plasmid psiCHECK2 (Promega - C8021) to generate a Renilla luciferase / SCAP fusion mRNA. Huh7 cells were cultured in DMEM (Gibco - 10313021) medium containing 10% fetal bovine serum (Gibco - 10099141C), 1% glutamine (Gibco - 35050061), 1% non-essential amino acids (Gibco - 11140050), and 1% penicillin-streptomycin (Gibco - 15070063). The SCAP-psiCHECK2 plasmid, RNAi agent, and Lipo2000 (Invitrogen - 11668019) transfection reagent diluted with Opti-MEM (Gibico - 11058021) were added to the Huh7 cell suspension, and plated in a 96-well plate at a cell density of 1*10 5 / mL, such that the final concentration of the RNAi agent was 1 nM and 0.02 nM. After culturing for 24 hours, the relative level of Renilla luciferase normalized to the level of constitutively expressed firefly luciferase also present on the psiCHECK2 plasmid was measured using the dual-luciferase reporter assay (Promega - E2920).

[0138] The inhibition rate of the SCAP gene mediated by the RNAi agent was calculated according to the following formula. SCAP gene inhibition rate (%) = (1 - relative level of Renilla luciferase in the sample / relative level of Renilla luciferase in the control group) × 100. The calculated SCAP inhibition rate was obtained.

[0139] The test results are shown in Tables 4 and 5 below.

[0140] Table 4

[0141]

[0142]

[0143] Table 5

[0144] Number Inhibition Rate % - 1nm Number Inhibition Rate % - 1nm S354M A S669M A S356M A S681M A S359M A S690M A S361M A S725M A S362M A S726M A S364M A S728M A S443M A S853M A S476M A S948M A S477M A S975M A S478M A S986M A S488M A S987M A S497M A S988M A S498M A S989M A S501M A S990M A S502M A S995M A S503M A S997M A S504M A S998M A S507M A S1003M A S512M A S1004M A S519M A S1010M A S537M A S1015M A S646M A S1030M A S668M A S1031M A

[0145]

[0146] Among them, inhibition rate %: A > 70%.

[0147] Example 8

[0148] In vitro test of SCAP RNAi agent in Huh7 cells. The exon region (including 5'UTR and 3'UTR sequences) of the human SCAP (NM_012235) gene was cloned into the reporter-based screening plasmid psiCHECK2 (Promega - C8021) to generate a Renilla luciferase / SCAP fusion mRNA. Huh7 cells were cultured in DMEM (Gibco - 10313021) medium containing 10% fetal bovine serum (Gibco - 10099141C), 1% glutamine (Gibco - 35050061), 1% non-essential amino acids (Gibco - 11140050), and 1% penicillin-streptomycin (Gibco - 15070063). The SCAP-psiCHECK2 plasmid, RNAi agent, and Lipo2000 (Invitrogen - 11668019) transfection reagent diluted with Opti-MEM (Gibico - 11058021) were added to the Huh7 cell suspension, and plated in a 96-well plate at a cell density of 1*10 5 / mL, such that the final concentrations of the RNAi agent were 1 nM and 0.02 nM. After culturing for 24 hours, the relative level of Renilla luciferase normalized to the level of constitutively expressed firefly luciferase also present on the psiCHECK2 plasmid was measured using the dual-luciferase reporter assay (Promega - E2920).

[0149] The inhibition rate of the SCAP gene mediated by the RNAi agent was calculated according to the following formula. SCAP gene inhibition rate (%) = (1 - relative level of Renilla luciferase in the sample / relative level of Renilla luciferase in the control group) × 100. The obtained SCAP inhibition rate was calculated.

[0150] The test results are shown in Table 6 below.

[0151] Table 6

[0152]

[0153] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An RNAi agent for inhibiting the expression of the SCAP gene, characterized in that, The RNAi agent includes a sense strand and an antisense strand, wherein the sense strand and the antisense strand are at least partially complementary, and the sense strand and the antisense strand are selected from the sequences shown in Table 1.

2. The RNAi agent according to claim 1, wherein The sense strand and / or the antisense strand contains at least one modified nucleotide.

3. The RNAi agent according to claim 1, wherein The modified nucleotide is selected from: alkyl nucleotide, methoxy nucleotide, ethoxy nucleotide, methoxyethyl nucleotide, amino nucleotide, fluoro nucleotide, deoxy nucleotide, 5'-methyl phosphate nucleotide, 5'-C-methyl phosphate nucleotide, 2'-deoxy-2'-fluoro nucleotide, vinyl phosphonate deoxyribonucleotide (VP), phosphorothioate nucleotide, dithiophosphate nucleotide, locked nucleic acid (LNA), morpholino oligonucleotide (PMO), glycol nucleic acid (GNA).

4. The RNAi agent according to claim 2 or 3, wherein The modified RNAi agent is selected from the sequences shown in Table 2.

5. The RNAi agent according to claim 1, wherein The RNAi agent further includes a targeting ligand, and the targeting ligand is linked to the sense strand and / or the antisense strand.

6. The RNAi agent according to claim 5, wherein The targeting ligand includes an N-acetyl-galactosamine (GalNAc) moiety.

7. The RNAi agent according to claim 5, wherein The targeting ligand is linked to the 3' or 5' end of the sense strand or the antisense strand.

8. The RNAi agent according to any one of claims 5-7, characterized in that, The targeting ligand is selected from: Among them, represents the siRNA ligation site, R 2 represents siRNA, and Nu represents siRNA.

9. A pharmaceutical composition for inhibiting the expression of SCAP gene, characterized in that, The pharmaceutical composition contains the RNA agent according to any one of claims 1-8.

10. Use of the RNAi agent according to any one of claims 1-8 in the preparation for treating SCAP gene-related diseases, disorders or symptoms, and the diseases are selected from treating elevated blood lipid levels and the like.

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