Sirna for inhibiting LPA gene expression, and modifier and use thereof

Abstract

A siRNA for inhibiting LPA gene expression, and a modifier and the use thereof, belonging to the field of biomedicine. Provided are a double-stranded RNA molecule targeting an LPA gene, and a modifier thereof. Both in vivo and in vitro experiments show that the provided double-stranded RNA molecule can effectively inhibit LPA gene expression, and can be used for preventing and / or treating Lp(a)-related diseases, such as thrombosis, atherosclerotic thrombosis, coronary artery disease, ischemic strokes, aortic valve stenosis, heart failure, atrial fibrillation, and peripheral artery disease.

Description

siRNA for inhibiting LPA gene expression, modification thereof and application TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to siRNA for inhibiting LPA gene expression, modification thereof and application. BACKGROUND

[0002] Lipoprotein(a) (Lp(a)) is formed by covalent binding of one molecule of apolipoprotein B100 (ApoB) and one molecule of apolipoprotein a [Apo(a)] by disulfide bond. Among them, the Apo(a) molecule contains the repeating structure of the fourth kringle domain of plasminogen (plasminogen kringle domain IV, abbreviated as KIV); KIV forms 10 KIV subtypes (named KIV-1 to KIV-10) through repeated expansion and differentiation. The number of KIV repeats is different among each subtype, and the size of Apo(a) protein is thus highly heterogeneous.

[0003] The plasma Lp(a) concentration is negatively correlated with the Apo(a) isoform. Isoform variation is caused by different numbers of kringle IV repeats in Apo(a). Different numbers of kringle units cause different Lp(a) levels in the general population. Generally speaking, the fewer the KIV repeats, the smaller the Lp(a) molecular weight, the smaller the protein particles, and the higher the Lp(a) level in serum; the more KIV repeats, the larger the Lp(a) molecular weight, the more accumulation of aop(a) precursors in the endoplasmic reticulum, and the lower the plasma Lp(a) concentration.

[0004] The normal Lp(a) level ranges from 0.1 to 25 mg / dl, and about 25% of the American population has an Lp(a) level of 30 mg / dl or higher. The American College of Cardiology (ACC) / American Heart Association (AHA) clinical practice guidelines suggest that Lp(a) > 50 mg / dL (125 nmol / L) increases the risk of cardiovascular disease; the European Atherosclerosis Society (EAS) suggests that Lp(a) at 30-50 mg / dL or 75-125 nmol / L should be considered in combination with other risk factors for further risk analysis.

[0005] Studies suggest that high levels of Lp(a) may promote the activation and aggregation of platelets, and are related to various thrombosis mechanisms; Lp(a) may promote the development of vulnerable plaques and promote atherosclerotic thrombosis, and is an independent risk factor for coronary artery disease, ischemic stroke, aortic valve stenosis, heart failure, atrial fibrillation and peripheral artery disease.

[0006] Lp(a) expression is limited to humans and non-human primates. Since Lp(a) levels in humans are genetically determined, Lp(a) levels cannot be directly reduced by lifestyle intervention alone, and the existing traditional lipid-lowering therapy cannot reduce Lp(a) to a level that is therapeutically significantly beneficial. SUMMARY

[0007] The technical problem to be solved by the present application is how to effectively regulate the abnormal expression of Lp(a), and more specifically, how to effectively inhibit the abnormal expression of Lp(a).

[0008] To solve the above technical problem, the present application provides a double-stranded RNA molecule, which can be any one of 274 siRNAs, the siRNA comprising a sense strand and an antisense strand forming at least a double-stranded region, the sense strand comprising a nucleotide sequence which can be any odd-numbered sequence in sequences 1-548, and the antisense strand comprising a nucleotide sequence which can be any even-numbered sequence in sequences 1-548.

[0009] Further, the length of the double-stranded region of the double-stranded RNA molecule can be 15-30 bp, 23-27 bp, 21-23 bp, 19-21 bp, 17-25 bp, 17-23 bp, 17-19 bp or 19 bp.

[0010] Further, the length of the sense strand of the double-stranded RNA molecule is not more than 30 nucleotides, and / or the length of the antisense strand is not more than 30 nucleotides.

[0011] Further, at least one of the sense strand and the antisense strand of the double-stranded RNA molecule comprises a 3' overhang having at least 1 nucleotide, or at least one of the sense strand and the antisense strand comprises a 3' overhang having at least 2 nucleotides.

[0012] Further, in the double-stranded RNA molecule, the nucleotide sequence of the sense strand of the 274 siRNAs can be numbered as n, the nucleotide sequence of the antisense strand of the 274 siRNAs can be numbered as n+1, and n can be any odd number from 1 to 548. The nucleotide sequence of the sense strand of the 274 siRNAs also includes a sequence having 90% or more identity to any of the odd-numbered sequences in sequences 1-548; and the nucleotide sequence of the antisense strand of the 274 siRNAs also includes a sequence having 90% or more identity to any of the even-numbered sequences in sequences 1-548.

[0013] The present application also provides a double-stranded RNA molecule modifier, which can be a compound containing a modified nucleotide obtained by modifying at least one nucleotide of the double-stranded RNA molecule.

[0014] The double-stranded RNA molecule modifier can be a compound containing modified nucleotides obtained by modifying at least one nucleotide of the double-stranded RNA molecule described above, and the sense strand of the double-stranded RNA molecule can be a nucleotide sequence that is any of the odd-numbered sequences in SEQ ID NO: 1-548.

[0015] Further, at least one nucleotide in the sense strand or the antisense strand of the double-stranded RNA molecule modifier can be a modified nucleotide.

[0016] Further, the modified nucleotide in the double-stranded RNA molecule modifier is selected from at least one of the following: 2'-methoxy-modified nucleotide, 2'-fluoro-modified nucleotide, deoxy-nucleotide, 2'-deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, non-locked nucleotide, configuration-restricted nucleotide, restricted ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxyl-modified nucleotide, 2'-methoxyethyl-modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, nucleotide containing a non-natural base, tetrahydropyran-modified nucleotide, 1,5-anhydrohexitol-modified nucleotide, cyclohexenyl-modified nucleotide, glycerol nucleotide (GNA), unlinked nucleotide (UNA), nucleotide containing a methylphosphonate group, nucleotide containing a 5'-phosphate, and nucleotide containing a 5'-phosphate mimic.

[0017] Further, the double-stranded RNA molecule modifier contains 2'-methoxy-modified nucleotides, which are located in the antisense strand and the sense strand of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, the 1st-6th, 10th-19th nucleotides of the sense strand can be 2'-methoxy-modified nucleotides; the 1st, 3rd-5th, 7th-13th, 15th, 17th-21st nucleotides of the antisense strand can be 2'-methoxy-modified nucleotides.

[0018] Further, the double-stranded RNA molecule modifier contains 2'-fluoro-modified nucleotides, which are located in the antisense strand and the sense strand of the double-stranded RNA molecule modifier, and, in the direction from the 5' end to the 3' end, the 7th, 8th, 9th nucleotides of the sense strand can be 2'-fluoro-modified nucleotides, and the 2nd, 6th, 14th, 16th nucleotides of the antisense strand can be 2'-fluoro-modified nucleotides.

[0019] Further, the double-stranded RNA molecule modifier contains at least one phosphorothioate-modified backbone.

[0020] Further, the phosphorothioate modification is located in the antisense strand and the sense strand of the double-stranded RNA molecule modifier, and, in the direction from 5' end to 3' end, the nucleotides at least at the first and the second position, the second and the third position of the sense strand are connected by phosphorothioate, and the nucleotides at least at the first and the second position, the second and the third position, the 19th and the 20th position, the 20th and the 21st position of the antisense strand are connected by phosphorothioate.

[0021] Further, in the double-stranded RNA molecule modifier, the ligand is connected to any nucleotide at the 3'-end or the 5'-end of the sense strand or in the middle of the sense strand.

[0022] Further, in the double-stranded RNA molecule modifier, the ligand can be a small molecule, an antibody, a polypeptide, a protein or an aptamer.

[0023] Further, in the double-stranded RNA molecule modifier, the small molecule can be L96.

[0024] Further, in the double-stranded RNA molecule modifier, the protein can be albumin.

[0025] Further, in the double-stranded RNA molecule modifier, the ligand is connected to the double-stranded RNA molecule modifier through a Linker.

[0026] The present application also provides the use of the double-stranded RNA molecule or the double-stranded RNA molecule modifier in any of the following:

[0027] D1) the use in the preparation of a composition for inhibiting the expression of LPA gene;

[0028] D2) the use in inhibiting the expression of LPA gene;

[0029] D3) the use in treating a disease related to LPA gene target;

[0030] D4) the use in the preparation of a composition for treating a disease related to LPA gene target.

[0031] The present application also provides a composition for inhibiting the expression of LPA gene, wherein the effective component can be the double-stranded RNA molecule or the double-stranded RNA molecule modifier.

[0032] Further, the composition further comprises a pharmaceutically acceptable carrier.

[0033] The composition can be a pharmaceutical composition or a kit.

[0034] The pharmaceutical composition described above, further comprising an unbuffered solution.

[0035] The unbuffered solution in the pharmaceutical composition described above can be normal saline or water.

[0036] The pharmaceutical composition described above, further comprising a buffered solution.

[0037] The buffered solution in the pharmaceutical composition described above comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

[0038] The buffered solution in the pharmaceutical composition described above can be phosphate buffered normal saline (PBS).

[0039] The present application also provides a cell comprising the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition.

[0040] The present application also provides a method of treating a LPA gene target-related disease, the method comprising administering the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition to a subject suffering from a LPA gene target-related disease.

[0041] The present application also provides a method of preventing and / or treating a thrombus, the method comprising administering the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition to a subject suffering from a thrombus.

[0042] The present application also provides a method of preventing and / or treating an ischemic stroke, the method comprising administering the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition to a subject suffering from an ischemic stroke.

[0043] The present application also provides a method of inhibiting LPA gene expression in a cell, the method comprising:

[0044] (a) contacting the cell with the double-stranded RNA molecule, the double-stranded RNA molecule modification, and / or the composition,

[0045] (b) maintaining the cell resulting from step (a) for a time sufficient to achieve degradation of the mRNA transcript of the LPA gene to effect inhibition of LPA gene expression in the cell.

[0046] Further, the method wherein the cell is located within a subject.

[0047] Further, the method wherein the subject can be a human.

[0048] Further, the method wherein the subject suffers from a Lp(a)-related disease.

[0049] Further, the method wherein the LPA gene expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100%.

[0050] As used herein, a "subject" can be an animal, such as a mammal, including a primate (e.g., a human or non-human primate, such as a monkey or chimpanzee). The "subject" can also be a transgenic animal that has been engineered to express human genes.

[0051] As used herein, the Lp(a) gene target related disease can be a disease caused by abnormal LPA gene expression.

[0052] As used herein, the Lp(a) gene target related disease can be a disease caused by abnormal LPA gene expression.

[0053] As used herein, the LPA gene target related disease can be a disease caused by abnormal LPA gene expression.

[0054] In one embodiment of the present application, the 2'-methoxy (2'-OMe) modified nucleotide, such as shown in formula (2), i.e., methoxy modification; 2'-substituted alkoxy modified nucleotide, such as 2'-O-methoxyethyl (2'-MOE) modified nucleotide, such as shown in formula (3), 2'-amino (2'-NH2) modified nucleotide, such as shown in formula (4), 2'-deoxynucleotide (DNA), such as shown in formula (5), wherein Base represents a modified or unmodified nucleotide base A, U, G, C, T or other nucleotide base.

[0055] In one embodiment of the present application, the nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide. The nucleotide analog can be an isonucleotide, a bridged nucleotide, or an acyclic nucleotide.

[0056] In an embodiment of the present application, the bridged nucleotide (BNA) refers to a constrained or inaccessible nucleotide, which can contain a five-, six-, or seven-membered ring bridged structure with a "fixed" C3'-endo sugar conformation, and the bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide.

[0057] In an embodiment of the present application, the BNA can be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8), wherein Base represents a modified or unmodified nucleotide base A, U, G, C, T or other nucleotide base.

[0058] In an embodiment of the present application, at least a portion of the phosphate group in the phosphor-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA is a phosphate group with a modification group.

[0059] In an embodiment of the present application, the phosphate group with a modification group is a phosphorothioate in which at least one oxygen atom in the phosphor diester bond of the phosphate group is replaced by a sulfur atom.

[0060] In an embodiment of the present application, the phosphate group with a modification group is a phosphorothioate with a structure shown in formula (9). In an embodiment of the present application, the phosphorothioate-linked nucleotide is shown in formula (10), and the phosphorothioate linkage exists in at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof.

[0061] In an embodiment of the present application, the VP-modified nucleotide is a vinyl phosphate modification. In an embodiment of the present application, the nucleotide modified by VP and methoxy modification, i.e., 5'-(E)-vinyl-2'-methoxy phosphonate (5'-(E)-VP-2'-OMe) modified nucleotide, is shown in formula (11); wherein Base represents a modified or unmodified nucleotide base A, U, G, C, T or other nucleotide base.

[0062] The nucleoside monomers used in the preparation of siRNA according to the present application are, unless otherwise specified, modified or unmodified nucleoside phosphoramidites (sometimes also referred to as RNA phosphoramidites) used in the phosphoramidite solid phase synthesis according to the kind and order of nucleotides in the siRNA to be prepared. The phosphoramidite solid phase synthesis is a method well known to the person skilled in the art for the synthesis of siRNA. All nucleoside monomers used according to the present application are commercially available.

[0063] In the present application, the sense strand of the double stranded RNA molecule is conjugated with a ligand attached at the 3'-end, and wherein the ligand is one or more GalNAc derivatives attached with a divalent or trivalent branched linker, such as L96.

[0064] The siRNA conjugate formed by L96 and the double stranded RNA molecule according to the present application has the following structure.

[0065] It is worth mentioning that all raw materials used in the present application are ordinary commercial products, and their sources are not specifically limited.

[0066] The present application also relates to the following technical solutions:

[0067] 1. A double stranded RNA molecule, wherein the double stranded RNA molecule comprises a sense strand and an antisense strand forming at least in part a double stranded region, the antisense strand comprising 17-21 consecutive nucleotides of the nucleotide sequence of any even-numbered SEQ ID NO. 1-548, and the sense strand comprising 17-19 consecutive nucleotides of the nucleotide sequence of any odd-numbered SEQ ID NO. 1-548.

[0068] 2. The double stranded RNA molecule according to item 1, wherein the length of the sense strand is no more than 30 nucleotides, and / or the length of the antisense strand is no more than 30 nucleotides, preferably the length of the sense strand is no more than 21 nucleotides, and / or the length of the antisense strand is no more than 23 nucleotides, more preferably the length of the sense strand is no more than 19 nucleotides, and / or the length of the antisense strand is no more than 21 nucleotides.

[0069] 3. The double stranded RNA molecule according to item 1 or 2, wherein the sense strand and the antisense strand of the double stranded RNA molecule comprise or are any combination selected from the following:

[0070] the sense strand of nucleotide sequence number n and the antisense strand of nucleotide sequence number n+1, wherein n is an odd number from 1 to 548.

[0071] 4. A modified double stranded RNA molecule comprising the double stranded RNA molecule of any one of items 1 to 3, and wherein at least one nucleotide is chemically modified, preferably the chemical modification comprises substitution of the ribosyl 2' position hydroxyl group of the nucleotide with another group, and / or modification of the base on the nucleotide, and / or substitution of the ribosyl 5' position hydroxyl group of the nucleotide with another group, and / or modification of the phosphate, further preferably the chemically modified nucleotide is selected from at least one of the group consisting of: 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, 5'-(E)-vinylphosphonate modified nucleotide, 3'-phosphorothioate modified nucleotide, 2'-methyl modified nucleotide, deoxy-nucleotide, 2'-deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide (such as LNA), unlocked nucleotide, conformationally restricted nucleotide, restricted ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-0-allyl-modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-0-alkyl-modified nucleotide, morpholino nucleotide, nucleotide comprising a non-natural base, tetrahydropyrane modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, glycerol nucleotide (GNA), unlocked nucleotide (UNA), nucleotide comprising a methylphosphonate group, nucleotide comprising a 5'-phosphate, and nucleotide comprising a 5'-phosphate mimic, more preferably the chemically modified nucleotide is selected from at least one of the group consisting of: locked nucleotide (such as LNA), 2'-deoxy-nucleotide, 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, 5'-(E)-vinylphosphonate modified nucleotide, 3'-phosphorothioate modified nucleotide.

[0072] 5. The modified double stranded RNA molecule according to item 4, optionally the 5' end and / or the 3' end of the sense strand is linked to an inverted abasic nucleotide (invab).

[0073] 6. The modified double stranded RNA molecule according to item 4 or 5, wherein the double stranded RNA molecule is selected from any one of the following:

[0074] (1) the sense strand is: UmsUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 549),

[0075] AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550);

[0076] (2) sense strand is:

[0077] (invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 551),

[0078] AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550);

[0079] (3) sense strand is:

[0080] (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552),

[0081] AmsGfsAmsUmGmAmCmCmAmAmGmCfUmUfGmGfCmAmAmGmsUm (SEQ ID NO. 553);

[0082] (4) sense strand is:

[0083] (invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 551),

[0084] AmsGfsAmUmGmAfCmCmAmAmGmCfUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 554);

[0085] (5) sense strand is: UmsUmsGmCmCmAmAfGfC(d)UmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 555),

[0086] AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550);

[0087] (6) sense strand is: T(LNA)sUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 556),

[0088] antisense strand is: AmsGfsAmUmGmAfCmCfAfAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 557);

[0089] (7) sense strand is: UmsAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 558),

[0090] antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559);

[0091] (8) sense strand is: T(LNA)sAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 560),

[0092] antisense strand is: UmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 561);

[0093] (9) sense strand is:

[0094] (invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms(invab) (SEQ ID NO. 562),

[0095] antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559);

[0096] (10) sense strand is:

[0097] (invab) sGmUmUmAmUmCmGfAmGfGmCfAmCmAmUmUmCmUmCmCmAms(invab) (SEQ ID NO. 563),

[0098] antisense strand is: UmsGfsGmsAmGmAmAmUmGmUmGmCfCmUfCmGfAmUmAmAmsCm (SEQ ID NO. 564);

[0099] (11) the sense strand is:

[0100] (Invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms (Invab) (SEQ ID NO. 562),

[0101] the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCfCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 565);

[0102] (12) the sense strand is: UmsAmsUmCmGmAmGfGfC(d)AmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 566),

[0103] the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559);

[0104] (13) the sense strand is: UmsUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 549),

[0105] the antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567);

[0106] (14) the sense strand is:

[0107] (invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 551),

[0108] the antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567);

[0109] (15) the sense strand is:

[0110] (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552),

[0111] The antisense strand is: VPAmsGfsAmsUmGmAmCmCmAmAmGmCfUmUfGmGfCmAmAmGmsUm (SEQ ID NO. 568);

[0112] (16) the sense strand is:

[0113] (Invab)sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (Invab) (SEQ ID NO. 551),

[0114] The antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCfUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 569);

[0115] (17) the sense strand is: UmsUmsGmCmCmAmAfGfC(d)UmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 555),

[0116] The antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567);

[0117] (18) the sense strand is: T(LNA)sUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 556),

[0118] The antisense strand is: VPAmsGfsAmUmGmAfCmCfAfAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 570);

[0119] (19) the sense strand is: UmsAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 558),

[0120] The antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 571);

[0121] (20) the sense strand is: T(LNA)sAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 560),

[0122] the antisense strand is: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572);

[0123] (21) the sense strand is:

[0124] (invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms (invab) (SEQ ID NO. 562),

[0125] the antisense strand is: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572);

[0126] (22) the sense strand is:

[0127] (invab) sGmUmUmAmUmCmGfAmGfGmCfAmCmAmUmUmCmUmCmCmAms (invab) (SEQ ID NO. 563),

[0128] the antisense strand is: VPUmsGfsGmsAmGmAmAmUmGmUmGmCfCmUfCmGfAmUmAmAmsCm (SEQ ID NO. 573);

[0129] (23) the sense strand is:

[0130] (invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms (invab) (SEQ ID NO. 562),

[0131] the antisense strand is: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572);

[0132] (24) the sense strand is: UmsAmsUmCmGmAmGfGfC(d)AmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 566),

[0133] the antisense strand is: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572);

[0134] wherein Af represents 2'-fluoro-adenosine-3'-phosphate, Afs represents 2'-fluoro- adenosine-3'-phosphorothioate, Am represents 2'-methoxy-adenosine-3'-phosphate, Ams represents 2'-methoxy-adenosine-3'-phosphorothioate, Cf represents 2'-fluoro- cytidine-3'-phosphate, Cfs represents 2'-fluoro-cytidine-3'-phosphorothioate, Cm represents 2'-methoxy-cytidine-3'-phosphate, Cms represents 2'-methoxy-cytidine-3'- phosphorothioate, Gf represents 2'-fluoro-guanosine-3'-phosphate, Gfs represents 2'- fluoro-guanosine-3'-phosphorothioate, Gm represents 2'-methoxy-guanosine-3'- phosphate, Gms represents 2'-methoxy-guanosine-3'-phosphorothioate, Uf represents 2'-fluoro-uridine-3'-phosphate, Ufs represents 2'-fluoro-uridine-3'- phosphorothioate, Um represents 2'-methoxy-uridine-3'-phosphate, Ums represents 2'- methoxy-uridine-3'-phosphorothioate, T(LNA)s represents C(d) represents 2' deoxy-cytidine-3' phosphate, (invab) at the 5' end represents: (invab) at the 3' end represents: VP represents a hydroxyl group at the 5' position of a nucleotide is replaced by an (E)-ethenyl phosphate.

[0135] 7. The modified double stranded RNA molecule according to any one of items 4-6, wherein the 3'-end of the sense strand is coupled to a ligand comprising N-acetylgalactosamine (GalNAc) or a derivative thereof, preferably the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, trivalent or tetravalent branched linker.

[0136] 8. The modified double stranded RNA molecule according to item 7, wherein the ligand comprises a compound according to the formula:

[0137] preferably the ligand is selected from a compound according to the formula:

[0138] the compound is coupled to the sense strand via a phosphate or phosphorothioate.

[0139] 9. A dimeric siRNA comprising the double stranded RNA molecule according to any one of items 1-3 or the modified double stranded RNA molecule according to any one of items 4-8.

[0140] 10. A pharmaceutical composition comprising the double stranded RNA molecule according to any one of items 1-3 or the modified double stranded RNA molecule according to any one of items 4-8 or the dimeric siRNA according to item 9, and a pharmaceutically acceptable carrier.

[0141] 11. Use of the double-stranded RNA molecule of any one of items 1 to 3 or the modified double-stranded RNA molecule of any one of items 4 to 8 or the duplex siRNA of item 9 or the pharmaceutical composition of item 10 in any one of:

[0142] D1) the manufacture of a composition for inhibiting expression of an LPA gene;

[0143] D2) inhibiting expression of an LPA gene;

[0144] D3) treating a disease associated with an LPA gene target;

[0145] D4) the manufacture of a composition for treating a disease associated with an LPA gene target.

[0146] 12. A method of treating a disease associated with an LPA gene target comprising administering to a subject a therapeutically effective amount of the double-stranded RNA molecule of any one of items 1 to 3 or the modified double-stranded RNA molecule of any one of items 4 to 8 or the duplex siRNA of item 9 or the pharmaceutical composition of item 10.

[0147] 13. A method of inhibiting expression of an LPA gene in a cell, the method comprising:

[0148] contacting the cell with the double-stranded RNA molecule of any one of items 1 to 3 or the modified double-stranded RNA molecule of any one of items 4 to 8 or the duplex siRNA of item 9 or the pharmaceutical composition of item 10; and maintaining the cell for a period of time sufficient to achieve degradation of the mRNA transcript of the LPA gene to inhibit expression of the LPA gene in the cell;

[0149] Preferably, the cell is in a subject or in vitro.

[0150] More preferably, the subject is afflicted with a disease associated with an LPA gene target.

[0151] 14. The use according to item 11 or the method of item 12 or 13, wherein the disease associated with an LPA gene target is selected from one or more of the group consisting of:

[0152] Berger's disease, peripheral arterial disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, ischemic stroke, heart failure, atrial fibrillation, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis.

[0153] 15. Use of a double stranded RNA molecule according to any one of items 1 to 3 or a modified double stranded RNA molecule according to any one of items 4 to 8 for the manufacture of a dimeric siRNA.

[0154] The present application provides a novel small interfering ribonucleic acid (siRNA) inhibitor of lipoprotein a [Lp(a)] and its use. Specifically, the present application provides siRNA that can be used as an Lp(a) inhibitor. The present application also relates to the LPA siRNA drug, and pharmaceutical compositions containing the siRNA. The siRNA inhibitor, by specifically recognizing and binding to LPA mRNA, causes RNA-induced silencing complex (RISC) to specifically degrade LPA mRNA, resulting in down-regulation of Lp(a) levels, and can be used as a long-term stable defense and effective therapeutic drug for Lp(a)-related diseases, including but not limited to Berger's disease, peripheral arterial disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis. The LPA small interfering RNA and the additional therapeutic drug can be administered in a single composition, or they can be administered separately. Examples of the additional therapeutic agent include, but are not limited to, HMg Co-A reductase inhibitors (statins), ezetimibe, PCSK-9 inhibitors, CTEP inhibitors, therapies targeting ANGPTL3, therapies targeting APOC3, and niacin, etc.

[0155] Innovation of the present application: Based on the above, the present application designs and screens a novel siRNA inhibitor that can efficiently degrade LPA mRNA and significantly reduce plasma Lp(a) levels, with the hope of achieving a zero breakthrough in Lp(a) target therapy drugs for cardiovascular diseases, and realizing long-term stable defense and effective treatment of Lp(a)-related cardiovascular diseases.

[0156] The LPA small interfering RNAs described herein can be used to prevent or treat, or to prepare a medicament for preventing or treating, a disease including, but not limited to, Berger's disease, peripheral arterial disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease, and venous thrombosis. The LPA small interfering RNA and the additional therapeutic agent can be administered in a single composition, or they can be administered separately.

[0157] Compared with the prior art, the application has the following beneficial technical effects:

[0158] The application provides novel double-stranded RNA molecules targeting LPA gene, and in vivo and in vitro experiments show that the double-stranded RNA molecules provided by the application can effectively inhibit LPA gene expression. It is shown that the double-stranded RNA molecules provided by the application have great potential for drug development and application value in the field of preventing and / or treating LPA gene expression abnormal diseases such as thrombosis, atherosclerotic thrombosis, coronary artery disease, ischemic stroke, aortic valve stenosis, heart failure, atrial fibrillation and peripheral arterial disease. DETAILED DESCRIPTION

[0159] The application will be further described below in conjunction with examples. It should be understood that the examples are only used to further illustrate and explain the application, and are not used to limit the application.

[0160] Definitions

[0161] In the application, "siRNA" is a kind of double-stranded RNA molecule, which can mediate the silencing of target RNA (for example, mRNA, for example, the transcript of a gene encoding a protein) complementary thereto. The siRNA includes an antisense strand complementary to the target RNA, and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such gene is also referred to as target gene. Generally, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, in addition to mRNA, RNA such as tRNA and viral RNA can also be targeted.

[0162] In the application, an "antisense strand" refers to a strand of an siRNA that comprises a region that is fully or substantially complementary to a target sequence. The term "region of complementarity" as used herein refers to a region on an antisense strand that is substantially complementary to a sequence (e.g., a target sequence). When a region of complementarity is not fully complementary to a target sequence, the mismatches are typically in the interior or terminal regions of the molecule. In some embodiments, a double-stranded nucleic acid molecule includes a nucleotide mismatch in the antisense strand.

[0163] In the application, the term "complementary" as used to describe a first nucleotide sequence with respect to a second nucleotide sequence means the ability of an oligonucleotide comprising the first nucleotide sequence to hybridize to and form a duplex structure with an oligonucleotide comprising the second nucleotide sequence under certain conditions, as understood by one of skill in the art.

[0164] In the application, the term "sense strand" refers to a strand of an siRNA that comprises a region that is substantially complementary to a region of an antisense strand as defined herein.

[0165] In the application, the term "overhang" refers to at least one unpaired nucleotide that protrudes from a duplex structure of an siRNA. For example, a nucleotide overhang exists when the 3 '-end of one strand of an siRNA extends beyond the 5 '-end of the other strand or vice versa. An siRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang can comprise or consist of nucleotides / nucleoside analogs, including deoxynucleotides / nucleosides. One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. Additionally, one or more nucleotides of an overhang can be present on the 5 '-end, the 3 '-end, or both ends of the antisense strand or the sense strand of an siRNA.

[0166] In the application, a region of complementarity within a double-stranded RNA molecule includes base pairing between the sense and antisense strands over the entire length. Such sequences can be referred to as "fully complementary" to one another in the application. However, when a sense strand is referred to as "substantially complementary" or "partially complementary" to an antisense strand herein, the two sequences can be fully complementary, or they can form one or more mismatched base pairs, such as 1, 2, 3, 4, or 5 mismatched base pairs, but preferably no more than 5, while retaining the ability to hybridize under conditions most relevant to their ultimate application. Overhangs should not be considered mismatches in the determination of complementarity. For example, a double-stranded RNA molecule comprising a sense strand of 19 nucleotides in length and an antisense strand of 21 nucleotides in length, wherein the longer nucleotide comprises a sequence of 19 nucleotides that is fully complementary to the shorter nucleotide, can still be referred to as "fully complementary."

[0167] In the present application, "modified nucleotide" includes a nucleotide or nucleotide analog in which the ribosyl 2' position hydroxyl group is replaced with another group, or a nucleotide or nucleotide analog in which the ribosyl 5' position hydroxyl group is replaced with another group, or a nucleotide in which the base is a modified base, or a nucleotide in which one of the oxygen atoms in the 3' phosphate is replaced with a sulfur atom to form a phosphorothioate. "Methoxy modified nucleotide" refers to a nucleotide in which the ribosyl 2'-hydroxyl group is replaced with a methoxy group. "Fluoro modified nucleotide" refers to a nucleotide in which the ribosyl 2' position hydroxyl group is replaced with a fluorine. "Nucleotide analog" refers to a group that can substitute for a nucleotide in a nucleic acid, but is structurally different from an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide.

[0168] In the present application, "ligand" refers to a chemical moiety conjugated to an siRNA that is capable of altering the distribution, targeting, or longevity of the siRNA. In preferred embodiments, such a ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cellular or organ compartment, a tissue, an organ, or a region of the body)) compared to, for example, an siRNA in the absence of such a ligand.

[0169] In the present application, Base represents a modified or unmodified nucleotide base A, U, G, C, T, or other nucleotide base.

[0170] In the present application, "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," and other similar terms, and includes any level of inhibition.

[0171] In the present application, "inhibit expression of LPA" refers to inhibiting expression of any LPA gene, as well as variants or mutants of LPA genes. Thus, the LPA gene can be a wild-type LPA gene, a mutant LPA gene, or a transgenic LPA gene in the context of a genetically manipulated cell, group of cells, or organism.

[0172] In the present application, "inhibit LPA gene expression" includes any level of inhibition of a LPA gene, such as at least partial suppression of LPA gene expression. LPA gene expression can be assessed based on the level or change in level of any variable associated with LPA gene expression, such as LPA mRNA level, LPA protein level, or lipid level. This level can be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

[0173] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with LPA expression compared to a control level. The control level can be any type of control level utilized in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., buffer control or inert agent control) treated subject, cell, or sample.

[0174] In the present application, "treatment" refers to beneficial or desired results including, but not limited to, alleviation or amelioration of one or more symptoms associated with gene expression. "Treatment" can also mean prolonging survival as compared to expected survival if not treated. Treatment can include prevention of the development of a complication, such as reducing liver injury in an individual having a liver infection.

[0175] In the present application, "therapeutically effective amount" is intended to include an amount of a nucleic acid (e.g., siRNA) that is sufficient to effect treatment (e.g., by reducing, ameliorating, or maintaining an existing disease, or one or more symptoms of a disease or its associated complications) when administered to a patient for treating a subject having a disease.

[0176] In the present application, "pharmaceutically acceptable" refers to those compounds, materials, compositions, or dosage forms which are suitable for use with humans and animal subjects without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio as in the art.

[0177] In the present application, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

[0178] Double-stranded RNA molecule

[0179] In one aspect, the present application provides an unmodified double-stranded RNA molecule, specifically an siRNA, wherein the double-stranded RNA molecule comprises a sense strand and an antisense strand forming at least in part a double-stranded region, the antisense strand comprising a contiguous 17-21 nucleotides, such as a contiguous 17, 18, 19, 20, or 21 nucleotides, of a nucleotide sequence of any even-numbered SEQ ID NO. 1-548, the sense strand comprising a contiguous 17-19 nucleotides, such as a contiguous 17, 18, or 19 nucleotides, of a nucleotide sequence of any odd-numbered SEQ ID NO. 1-548.

[0180] In some embodiments, the double-stranded region is 15-30 bp in length, e.g., can be 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp. In some embodiments, the double-stranded region is 17-27 bp in length. In some embodiments, the double-stranded region is 19-23 bp in length. In some embodiments, the double-stranded region is 19-21 bp in length. In some embodiments, the double-stranded region is 17-25 bp in length. In some embodiments, the double-stranded region is 17-23 bp in length. In some embodiments, the double-stranded region is 17-19 bp in length. In some embodiments, the double-stranded region is 19 bp, 20 bp, or 21 bp in length.

[0181] In some embodiments, the sense strand is no more than 30 nucleotides in length, and / or the antisense strand is no more than 30 nucleotides in length. In some embodiments, the sense strand is no more than 23 nucleotides in length, and / or the antisense strand is no more than 25 nucleotides in length. In some embodiments, the sense strand is no more than 19 nucleotides in length, and / or the antisense strand is no more than 21 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length, and / or the antisense strand is 21 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length, and / or the antisense strand is 21 nucleotides in length.

[0182] In some embodiments, the sense strand and the antisense strand are fully or partially complementary. In some embodiments, the complementary region is 15, 16, 17, 18, 19, 20, or 21 nucleotides in length.

[0183] In some embodiments, at least one of the sense strand and the antisense strand comprises a 3' overhang of at least 1 or at least 2 nucleotides.

[0184] In some embodiments, the sense strand of the double-stranded RNA molecule has the nucleotide sequence of any odd-numbered SEQ ID NO. 1-548, and the antisense strand has the nucleotide sequence of any even-numbered SEQ ID NO. 1-548.

[0185] In some embodiments, the sense strand and the antisense strand of the double-stranded RNA molecule comprise or are any combination of Table 1 herein.

[0186] In another aspect, the present application also provides a modified double-stranded RNA molecule, comprising any one of the above unmodified double-stranded RNA molecules, and wherein at least one nucleotide is chemically modified. That is, by modifying at least one nucleotide of any one of the above unmodified double-stranded RNA molecules to obtain a compound containing a modified nucleotide.

[0187] The chemical modification can be substitution of the ribosyl 2' position hydroxyl group of the nucleotide with another group, can be modification of the base on the nucleotide, can be substitution of the ribosyl 5' position hydroxyl group of the nucleotide with another group, can be modification of the phosphodiester between the nucleotides (i.e. modification of the 3' phosphate of the nucleotide), or can be any combination of these.

[0188] In some embodiments, the chemical modification comprises substitution of the ribosyl 2' position hydroxyl group of the nucleotide with another group, and / or modification of the base on the nucleotide, and / or substitution of the ribosyl 5' position hydroxyl group of the nucleotide with another group.

[0189] In some embodiments, the modified nucleotide is selected from at least one of the following group:

[0190] 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, 5'-(E)-vinyl phosphonate modified nucleotide, 3' phosphate modified nucleotide, 2'-methyl modified nucleotide, deoxy-nucleotide, 2'-deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide (LNA), unlocked nucleotide, conformationally restricted nucleotide, restricted ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, nucleotide comprising a non-natural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, glycerol nucleotide (GNA), unlocked nucleotide (UNA), nucleotide comprising a methylphosphonate group, nucleotide comprising a 5'-phosphate, and nucleotide comprising a 5'-phosphate mimic.

[0191] In some embodiments, the chemically modified nucleotide is selected from at least one of the following group: inverted abasic deoxyribose modified nucleotide, locked nucleotide (LNA), 2'-deoxy-nucleotide, 2'-methoxy modified nucleotide, 2'-fluoro modified nucleotide, 5'-(E)-vinyl phosphonate modified nucleotide.

[0192] In some embodiments, the chemical modification further comprises a modification to the phosphodiester between nucleotides (i.e. a modification to the phosphate group at the 3' position of a nucleotide). In some embodiments, the modification to the phosphodiester is a phosphorothioate modification.

[0193] In one embodiment of the present application, the 5' end and / or 3' end of the sense strand of the double-stranded RNA molecule is further linked with an inverted abasic nucleotide (invab).

[0194] In some embodiments, when the invab is linked at the 5' end of the sense strand, the structure is as follows: which can also be a phosphorothioate modification (invab) s: When the invab is linked at the 3' end of the sense strand, it is as follows: When L96 is coupled at the 3' end, it is as follows:

[0195] In one embodiment of the present application, the modified nucleotide is a 2'-deoxy modified nucleotide, such as C(d) representing 2' deoxy-cytidine-3' phosphate.

[0196] In one embodiment of the present application, the modified nucleotide is a locked nucleic acid (LNA), such as T(LNA) s as follows:

[0197] In some embodiments, the double-stranded RNA molecule is selected from any one of the following:

[0198] (1) the sense strand is: UmsUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 549),

[0199] the antisense strand is: AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550);

[0200] (2) the sense strand is:

[0201] (invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms(invab) (SEQ ID NO. 551),

[0202] the antisense strand is: AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550);

[0203] (3) the sense strand is:

[0204] (Invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (Invab) (SEQ ID NO. 551),

[0205] the antisense strand is: AmsGfsAmUmGmAfCmCmAmAmGmCfUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 554);

[0206] (4) the sense strand is:

[0207] (Invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (Invab) (SEQ ID NO. 551),

[0208] the antisense strand is: AmsGfsAmUmGmAfCmCmAmAmGmCfUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 554);

[0209] (5) the sense strand is: UmsUmsGmCmCmAmAfGfC(d)UmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 555),

[0210] the antisense strand is: AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550);

[0211] (6) the sense strand is: T(LNA) sUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 556),

[0212] the antisense strand is: AmsGfsAmUmGmAfCmCfAfAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 557);

[0213] (7) the sense strand is: UmsAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 558),

[0214] the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559);

[0215] (8) the sense strand is: T(LNA)sAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 560),

[0216] the antisense strand is: UmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 561);

[0217] (9) the sense strand is:

[0218] (invab)sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms(invab) (SEQ ID NO. 562),

[0219] the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559);

[0220] (10) the sense strand is:

[0221] (invab)sGmUmUmAmUmCmGfAmGfGmCfAmCmAmUmUmCmUmCmCmAms(invab) (SEQ ID NO. 563),

[0222] the antisense strand is: UmsGfsGmsAmGmAmAmUmGmUmGmCfCmUfCmGfAmUmAmAmsCm (SEQ ID NO. 564);

[0223] (11) the sense strand is:

[0224] (Invab)sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms(Invab) (SEQ ID NO. 562),

[0225] the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCfCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 565);

[0226] (12) the sense strand is: UmsAmsUmCmGmAmGfGfC(d)AmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 566),

[0227] UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559);

[0228] (13) sense strand is:

[0229] antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567);

[0230] (14) sense strand is:

[0231] (invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 551),

[0232] antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567);

[0233] (15) sense strand is:

[0234] (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552),

[0235] antisense strand is: VPAmsGfsAmsUmGmAmCmCmAmAmGmCfUmUfGmGfCmAmAmGmsUm (SEQ ID NO. 568);

[0236] (16) sense strand is:

[0237] (invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 551),

[0238] antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCfUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 569);

[0239] (17) sense: UmsUmsGmCmCmAmAfGfC(d)UmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 555),

[0240] antisense: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567);

[0241] (18) sense: T(LNA)sUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 556),

[0242] antisense: VPAmsGfsAmUmGmAfCmCfAfAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 570);

[0243] (19) sense: UmsAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 558),

[0244] antisense: VPUmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 571);

[0245] (20) sense: T(LNA)sAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 560),

[0246] antisense: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572);

[0247] (21) sense:

[0248] antisense: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572);

[0249] antisense: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572);

[0250] (22) sense:

[0251] (Invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms(Invab) (SEQ ID NO. 562),

[0252] the antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCfCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 574);

[0253] (23) the sense strand is:

[0254] (Invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms(Invab) (SEQ ID NO. 562),

[0255] the antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCfCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 574);

[0256] (24) the sense strand is: UmsAmsUmCmGmAmGfGfC(d)AmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 566),

[0257] the antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 571);

[0258] wherein Af represents 2’-fluoro adenosine-3’-phosphate, Afs represents 2’-fluoro adenosine-3’- phosphorothioate, Am represents 2’-methoxy adenosine-3’-phosphate, Ams represents 2’-methoxy adenosine-3’-phosphorothioate, Cf represents 2’-fluoro cytidine-3’-phosphate, Cfs represents 2’-fluoro cytidine-3’-phosphorothioate, Cm represents 2’-methoxy cytidine-3’-phosphate, Cms represents 2’-methoxy cytidine-3’-phosphorothioate, Gf represents 2’-fluoro guanosine-3’-phosphate, Gfs represents 2’-fluoro guanosine-3’-phosphorothioate, Gm represents 2’-methoxy guanosine-3’-phosphate, Gms represents 2’-methoxy guanosine-3’-phosphorothioate, Uf represents 2’-fluoro uridine-3’-phosphate, Ufs represents 2’-fluoro uridine-3’-phosphorothioate, Um represents 2’-methoxy uridine-3’-phosphate, Ums represents 2’-methoxy uridine-3’-phosphorothioate, T(LNA) s represents C(d) represents 2' deoxy-cytidine-3' phosphate, (invab) at the 5' end represents: (invab) at the 3' end represents: VP represents the hydroxyl group at the 5' position of the nucleotide is replaced by (E)-ethenyl phosphate.

[0259] The double-stranded RNA molecules of the present application, as well as the double-stranded RNA molecules conjugated with ligands, can be prepared by methods known in the art. For example, the phosphoramidite solid phase synthesis method known to those skilled in the art. The nucleotide monomers used in the present application are commercially available.

[0260] The unmodified or modified double-stranded RNA molecules of the present application can be further conjugated with ligands.

[0261] In some embodiments, the ligand can be conjugated to the 3'-end or 5'-end of the sense strand or any nucleotide in the middle of the sense strand. In some embodiments, the ligand is conjugated to the 3'-end of the sense strand.

[0262] In some embodiments, the ligand comprises a ligand of N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0263] In some embodiments, the ligand is one or more GalNAc derivatives attached by a monovalent, divalent, trivalent or tetravalent branched linker.

[0264] In some embodiments, the ligand is L96.

[0265] In some embodiments, the siRNA conjugate formed by the L96 and the siRNA molecule of the present application has the following structure:

[0266] Those skilled in the art can understand that the conjugation of the ligand to the sense strand can be direct conjugation or indirect conjugation through a linker. In some embodiments, the ligand is conjugated to the 3'-end of the sense strand through a linker. In some embodiments, the L96 is conjugated to the 3'-end of the sense strand through a phosphate or a phosphorothioate.

[0267] Dimeric siRNA

[0268] The present application also provides a dimeric siRNA comprising the unmodified double-stranded RNA molecule or the modified double-stranded RNA molecule described above.

[0269] The dimeric siRNA is obtained by covalently linking two siRNAs, which can selectively and effectively reduce or inhibit the expression of a target gene in a subject (e.g., a human or animal subject).

[0270] In some embodiments, the two siRNAs in the dimeric siRNA are covalently linked by the sense strands.

[0271] In some embodiments, the sense strands of the two siRNAs in the dimeric siRNA are covalently linked by a linker.

[0272] In some embodiments, one end of the linker is connected to the 3’ end of the sense strand of one siRNA and the other end is connected to the 5’ end of the sense strand of the other siRNA.

[0273] In some embodiments, the sense strands of the multimeric siRNA are conjugated to at least one ligand.

[0274] In some embodiments, the ligand comprises N-acetylgalactosamine (GalNAc) or a derivative thereof.

[0275] In some embodiments, the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, trivalent, or tetravalent branched linker.

[0276] In some embodiments, the ligand is L96.

[0277] In some embodiments, the ligand is conjugated to the 3’ end of the sense strand of one of the siRNAs.

[0278] The present application also provides a pharmaceutical composition comprising the unmodified double-stranded RNA molecule or the modified double-stranded RNA molecule or the dimeric siRNA described above, and a pharmaceutically acceptable carrier.

[0279] The pharmaceutically acceptable carrier can be an excipient, a stabilizer, a suspending agent, or a diluent, etc., which is well known to those skilled in the art.

[0280] The pharmaceutical composition of the present application can be administered at a dose sufficient to inhibit gene expression. Generally, a suitable dose of the double-stranded RNA molecule of the present application is about 0.001 to about 200.0 mg per kilogram of body weight of the subject per day, typically about 1 to 50 mg per kilogram of body weight of the subject per day. Generally, a suitable dose of the double-stranded RNA molecule of the present application is about 0.1 mg / kg to about 10 mg / kg, for example, about 1 mg / kg to about 5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg.

[0281] A repeated dosage regimen can include periodic (e.g., every other day or once a year) administration of a therapeutic amount of the nucleic acid. In certain embodiments, the frequency of administration of the nucleic acid (e.g., siRNA) is about once a month to about once a year, such as about once every three months, about once every half a year, about once every nine months.

[0282] In some embodiments, the double stranded RNA molecules of the present application are administered at a dose of about 0.1 mg / kg to about 5.0 mg / kg once about every week, once a month, once every two months, once every quarter (i.e., every three months), once every six months, once every nine months, once a year. In certain embodiments, the double stranded RNA molecules of the present application are administered to a subject once a week. In some embodiments, the double stranded RNA molecules of the present application are administered to a subject once a month. In some embodiments, the double stranded RNA molecules of the present application are administered once every quarter (i.e., every three months). In some embodiments, the double stranded RNA molecules of the present application are administered once every six months.

[0283] After the initial treatment regimen, the frequency of treatment can be reduced. For example, after administration once a week or once every two weeks for three months, administration can be repeated once a month for six months or a year; or longer.

[0284] The pharmaceutical compositions of the present application can be administered in a number of ways, in certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In some embodiments, the double stranded RNA molecules of the present application are administered subcutaneously to a subject.

[0285] Therapeutic methods and therapeutic uses

[0286] The present application also provides the use of the above unmodified double stranded RNA molecules or modified double stranded RNA molecules or duplex siRNAs or pharmaceutical compositions in any of the following:

[0287] D1) in the manufacture of a medicament for inhibiting expression of an LPA gene;

[0288] D2) for use in inhibiting expression of an LPA gene;

[0289] D3) for use in treating a disease associated with an LPA gene target;

[0290] D4) in the manufacture of a medicament for treating a disease associated with an LPA gene target.

[0291] Wherein, inhibiting expression of an LPA gene can mean inhibiting at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100%.

[0292] The disease associated with an LPA gene target can encompass a disease known in the art caused by abnormal expression of an LPA gene.

[0293] In some embodiments, the disease caused by abnormal expression of an LPA gene is a disease caused by upregulation of expression of an LPA gene.

[0294] In some embodiments, the LPA gene target related disease is selected from one or more of the following:

[0295] Berger's disease, peripheral arterial disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, ischemic stroke, heart failure, atrial fibrillation, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis.

[0296] The present application also provides a method for treating a LPA gene target related disease, comprising administering to a subject a therapeutically effective amount of an unmodified double-stranded RNA molecule or a modified double-stranded RNA molecule or a duplex siRNA, or a pharmaceutical composition as described above.

[0297] The present application also provides a method for inhibiting LPA gene expression in a cell, the method comprising:

[0298] contacting the cell with an unmodified double-stranded RNA molecule or a modified double-stranded RNA molecule or a duplex siNRA as described above, or a pharmaceutical composition as described above; and maintaining the cell for a time sufficient to achieve degradation of the mRNA transcript of the LPA gene to inhibit LPA gene expression in the cell.

[0299] In some embodiments, the cell is in a subject. In some embodiments, the cell is outside of a subject. The subject can be a mammal, including a primate (e.g., a human or a non-human primate, such as a monkey or a chimpanzee), a non-primate (e.g., a cow, a pig, a camel, an alpaca, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, or a whale). In some embodiments, the subject is a human.

[0300] In some embodiments, the subject is suffering from a LPA gene target related disease. The LPA gene target related disease is as described above.

[0301] Examples

[0302] The experimental methods in the following examples are routine methods, and are performed according to the techniques or conditions described in the literature in the art or according to the instructions of the products, unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0303] The quantitative tests in the following examples are performed in triplicate, and the results are averaged, unless otherwise specified.

[0304] The following examples use GraphPad Prism statistical software to process data, and the experimental results are expressed as mean ± standard deviation.

[0305] In the following examples, A represents adenosine-3'-phosphate (adenine ribonucleotide). Af represents 2'-fluoro adenosine-3'-phosphate (2'-fluoro-modified adenine ribonucleotide). Afs represents 2'-fluoro adenosine-3'-phosphorothioate (2'-fluoro-modified and 3'-phosphorothioate-modified adenine ribonucleotide). Am represents 2'-methoxy adenosine-3'-phosphate (2'-methoxy-modified adenine ribonucleotide). Ams represents 2'-methoxy adenosine-3'-phosphorothioate (2'-methoxy-modified and 3'-phosphorothioate-modified adenine ribonucleotide). C represents cytidine-3'-phosphate (cytosine ribonucleotide). Cf represents 2'-fluoro cytidine-3'-phosphate (2'-fluoro-modified cytosine ribonucleotide). Cfs represents 2'-fluoro cytidine-3'-phosphorothioate (2'-fluoro-modified and 3'-phosphorothioate-modified cytosine ribonucleotide). Cm represents 2'-methoxy cytidine-3'-phosphate (2'-methoxy-modified cytosine ribonucleotide). Cms represents 2'-methoxy cytidine-3'-phosphorothioate (2'-methoxy-modified and 3'-phosphorothioate-modified cytosine ribonucleotide). G represents guanosine-3'-phosphate (guanine ribonucleotide). Gf represents 2'-fluoro guanosine-3'-phosphate (2'-fluoro-modified guanine ribonucleotide). Gfs represents 2'-fluoro guanosine-3'-phosphorothioate (2'-fluoro-modified and 3'-phosphorothioate-modified guanine ribonucleotide). Gm represents 2'-methoxy guanosine-3'-phosphate (2'-methoxy-modified guanine ribonucleotide). Gms represents 2'-methoxy guanosine-3'-phosphorothioate (2'-methoxy-modified and 3'-phosphorothioate-modified guanine ribonucleotide). T represents thymidine-3'-phosphate (thymine deoxyribonucleotide). U represents uridine-3'-phosphate (uracil ribonucleotide). Uf represents 2'-fluoro uridine-3'-phosphate (2'-fluoro-modified uracil ribonucleotide). Ufs represents 2'-fluoro uridine-3'-phosphorothioate (2'-fluoro-modified and 3'-phosphorothioate-modified uracil ribonucleotide). Um represents 2'-methoxy uridine-3'-phosphate (2'-methoxy-modified uracil ribonucleotide). Ums represents 2'-methoxy uridine-3'-phosphorothioate (2'-methoxy-modified and 3'-phosphorothioate-modified uracil ribonucleotide). GalNAc represents N-acetylgalactosamine. VP represents (E)-vinyl phosphate, VPAms represents: VPUms represents: VPGms represents: VPCms represents: T(LNA)s represents C(d) represents 2' deoxy-cytidine-3' phosphate (invab) at the 5' end represents: (invab) at the 3' end represents:

[0306] Example 1, Double-stranded RNA molecules and modified double-stranded RNA molecule modifications thereof

[0307] hLPA mRNA refers to mRNA having the sequence shown in GeneBank registration number NM_005577.4, and the following siRNAs are all directed against NM_005577.4, and were synthesized by Suzhou Jimake Gene Co., Ltd. using a solid-phase synthesis method known in the art.

[0308] The unmodified siRNAs shown in Table 1 below were synthesized using the above-mentioned solid-phase synthesis method.

[0309] Unmodified siRNA sequences

[0310] Table 1. Sequences of the sense and antisense strands of unmodified siRNAs

[0311] Modified siRNA sequences

[0312] The siRNAs shown in Table 1 were modified to obtain modified siRNAs.

[0313] The modification modes include M1, M3, M6, AM3, J1, AM7, M17, M1VP, M3VP, M6VP, AM3VP, J1VP, AM7VP, M17VP, etc.

[0314] M1 modification mode:

[0315] Sense strand 5'-3'

[0316] ms-ms-m-m-m-m-f-f-f-m-m-m-m-m-m-m-m-m-m

[0317] i.e. the nucleotides at positions 1-6, 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 7-9 are 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2 and 2 and 3 are linked by phosphorothioate.

[0318] Antisense strand: 5 '-3 '

[0319] ms-fs-m-m-m-f-m-m-m-m-m-m-m-f-m-f-m-m-ms-ms-m

[0320] i.e. the nucleotides at positions 1, 3-5, 7-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, 16 are 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are linked by phosphorothioate.

[0321] According to the M1 modification mode, the unmodified siRNA in Table 1 is modified to obtain the M1 modified siRNA, and the modified siRNA is named as the corresponding naked sequence name after adding M1, for example, the siRNA number of Lpa-35 modified according to the M1 mode is Lpa-35M1.

[0322] On the basis of the M1 modification mode, 5'-(E)-VP modification is added at the 5' end of the antisense strand to obtain the siRNA modified according to the M1VP modification mode. M3 modification mode:

[0323] Antisense strand: 5 '-3 '

[0324] ms-fs-m-m-m-f-m-m-m-m-m-m-m-f-m-f-m-m-ms-ms-m

[0325] i.e. the nucleotides at positions 1-4, 6, 10-19 of the sense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 5, 7-9 are 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2 and 2 and 3 are linked by phosphorothioate.

[0326] Antisense strand: 5 '-3 '

[0327] ms-fs-m-m-m-f-m-m-m-m-m-m-m-f-m-f-m-m-ms-ms-m

[0328] The nucleotides at positions 1, 3-5, 7-13, 15, 17-21 of the antisense strand are 2'-methoxy modified nucleotides, the nucleotides at positions 2, 6, 14, 16 are 2'-fluoro modified nucleotides, and the nucleotides at positions 1 and 2, 2 and 3, 19 and 20, 20 and 21 are linked by phosphorothioate.

[0329] The unmodified siRNAs in Table 1 are modified according to the modification mode of M3 to obtain siRNAs modified according to the modification mode of M3, and the modified siRNAs are named by adding M3 after the corresponding naked sequence name. For example, the siRNA obtained by modifying Lpa-35 according to the modification mode of M3 is named Lpa-35M3.

[0330] Based on the modification mode of M3, 5'-(E)-VP modification is added to the 5' end of the antisense strand to obtain siRNAs modified according to the modification mode of M3VP. Similarly, the unmodified siRNAs in Table 1 are modified according to the modification modes of M6, AM3, J1, AM7, and M17 to obtain siRNAs modified according to the corresponding modification modes, and the modified siRNAs are named by adding M6, AM3, J1, AM7, or M17 after the corresponding naked sequence name.

[0331] Based on the modification modes of M6, AM3, J1, AM7, and M17, 5'-(E)-VP modification is added to the 5' end of the antisense strand to obtain siRNAs modified according to the modification modes of M6VP, AM3VP, J1VP, AM7VP, and M17VP.

[0332] The sequence information of the exemplary modified siRNAs is shown in Table 2 below.

[0333] Table 2

[0334] The siRNAs obtained by coupling L96 to the 3' end of the sense strand of the siRNAs modified according to the modification mode of M1 are named by adding M1G after the corresponding naked sequence number. For example, the siRNA obtained by coupling L96 to the siRNA modified according to the modification mode of M1 is named Lpa-35M1G. The siRNAs obtained by coupling L96 to the siRNAs modified according to the modification mode of M3 are named by adding M3G after the corresponding naked sequence number. For example, the siRNA obtained by coupling L96 to the siRNA modified according to the modification mode of M3 is named Lpa-35M3G.

[0335] Similarly, the siRNA obtained by coupling L96 to the 3' end of the sense strand of siRNA modified according to the M6, AM3, J1, AM7, M17 and the like is coupled L96 siRNA, and the corresponding siRNA number is: the corresponding naked sequence number M6, AM3, J1, AM7 or M17 is increased by G. For example, the siRNA obtained by coupling L96 to Lpa-558M6 is numbered Lpa-558M6G.

[0336] The siRNA obtained by coupling L96 to the 3' end of the sense strand of siRNA modified according to the M1VP, M3VP, M6VP, AM3VP, J1VP, AM7VP, M17VP and the like is coupled L96 siRNA, and the corresponding siRNA number is: the corresponding naked sequence number M1GVP, M3GVP, M6GVP, AM3GVP, J1GVP, AM7GVP or M17GVP is increased. For example, the siRNA obtained by coupling L96 to Lpa-558M6VP is numbered Lpa-558M6GVP.

[0337] The sequence information of the exemplary siRNA coupled L96 is shown in Table 3 below:

[0338] Table 3

[0339] Example 2, psiCHECK of M1 modified siRNA for inhibiting hLPA in target activity detection experiment

[0340] The psiCHECKTM-2 (Promega TM ) is used as a carrier to construct the plasmid psiCHECKTM-2-LPA, and the biological activity of LPA siRNA is detected, and thus the activity of LPA siRNA is preliminarily screened at the cellular level. The psiCHECKTM-2 vector uses Renilla luciferase as the main reporter gene, and the target gene fragment is cloned into the multiple cloning site downstream of the Renilla luciferase translation termination codon to form the psiCHECK2-LPA plasmid (see Table 4).

[0341] Table 4 Target sequence inserted in psiCHECK2-target gene plasmid

[0342] When the plasmid is introduced into cells, siRNA treatment is performed. If the siRNA binds to the target gene fragment, it will trigger the targeted degradation of the target gene, i.e. the RNAi process, resulting in the degradation of the Renilla luciferase mRNA. In this way, the degree of decrease in the activity of Renilla luciferase can be detected to determine whether there is a targeting relationship between siRNA and the target gene fragment, thereby achieving the preliminary screening of siRNA. The specific experimental process is as follows:

[0343] Step one: construction of detection plasmid hLPA-psiCHECK2

[0344] psiCHECK TM -2(PromegaTM) vector to construct 13 psiCHECK TM -2-LPA gene fragment plasmids, which contain the insertion sequences shown in SEQ ID NO: 2201-2213, are constructed into the Xho I / Not I site of the vector to obtain the detection plasmid hLPA-psiCHECK2; and are respectively named as: hLPA-psiCHECK2-1 to hLPA-psiCHECK2-13.

[0345] Step two: cell culture and transfection

[0346] Add 5 μL of siRNA of the corresponding concentration to each well of the 96-well plate, and then add 12.5 μL of Opti-MEM (containing the corresponding psiCHECK2 TM -hCFB plasmid 20 ng); then add 32.5 μL of Opti-MEM mixed with 0.3 μL of Lipofectamine2000 (purchased from Invitrogen, item number 11668-019) to each well. Mix well at room temperature and stand for 15 minutes. Add 50 μL of DMEM medium cell suspension containing 1×10 6 HEK293T cells to each well (DMEM complete medium, purchased from Transgen Biotech, item number FI101-01). Place in a 37°C incubator with 5% CO2 and 95% air for 24 hours for subsequent dual luciferase reporter gene system detection. Note:

[0347] The final concentration of siRNA is 1 nM, 0.1 nM or 0.01 nM;

[0348] Mock group: solvent control group, no siRNA is added, only an equal volume of DEPC (diethyl pyrocarbonate) water is added.

[0349] Step three: dual luciferase detection

[0350] Remove the cell plate and aspirate 20 μL of culture medium using the Bravo liquid handler. Add 75 μL / well of Luciferase Reagent (Promega, Cat. No. E2940) using the Bravo liquid handler. Place the plate on the shaker and incubate for 20 min at room temperature at 30 rpm. Mix the lysate well using the Bravo liquid handler and transfer 75 μL to the cell assay plate. Read the Firefly luminescence values using a multi-function plate reader. Add 75 μL of Stop&Glo Reagent to each well of the assay plate using the fully automated liquid handler. Detect the Renilla luminescence values using a multi-function plate reader.

[0351] Data processing: Normalize the Renilla luminescence values by the Firefly luminescence values in each well using the formula R = Renilla luminescence / Firefly luminescence. The luminescence ratio of each test group or control group is the average of the luminescence ratio of three culture wells. The inhibition activity of each siRNA on LPA mRNA is expressed as the inhibition percentage compared with the control group, inhibition rate % = (1 - R 测试组 / R mock ) x 100%. The experimental results are shown in Table 5 below.

[0352] Table 5. psiCHECK2 activity of modified siRNAs (Lpa-1M1 to Lpa-550M1) for inhibiting hLPA.

[0353] Example 3. Cell line activity assay for unmodified siRNAs for inhibiting hLPA

[0354] Step 1: Construction of hLPA stable cell line

[0355] For hLPA gene NM_005577.4, the PiggyBac (PB) transposon was used to insert a small fragment between EcoRI and XbaI of the PiggyBac (PB) transposon plasmid Y13345-PiggyBac-CMV-MCS-EF1a-Puro (Suzhou Jimma product, item number Y13345) replaced with the CDS sequence of hLPA gene NM_005577.4 to obtain the recombinant plasmid named hLPA PB-LPA-CDS_240517 plasmid. The PB-LPA-CDS_240517 plasmid hLPA PB plasmid and transposase plasmid (Suzhou Jimma product, Y13346) were co-transfected into 293T cells at a ratio of 10:1 (2ug:0.2ug). After 48 hours of transfection, puromycin (2ug / mL) (purchased from Invivo Gen, item number ant-pr) was used for screening until the cells were not dead. The cell strain was harvested, which was the 293T cell line stably transfected with hLPA CDS sequence, named 293T / hLPA, and qPCR detection was performed for verification. 293T / hLPA was used as a screening cell line.

[0356] Step two: cell culture and transfection

[0357] siRNA transfection was performed using transfection reagent Lipofectamine RNAiMAX. According to the instructions, the transfection complex was prepared. For example, to prepare a single-well transfection complex, siRNA was diluted with opti-MEM to prepare siRNA diluent, and 25uL of siRNA diluent was taken per well. 25uL of opti-MEM was mixed with 0.25uL of Lipofectamine RNAiMAX transfection reagent to prepare transfection reagent diluent. The above two diluents were mixed and gently mixed, and then incubated at room temperature for 10-20 minutes. The siRNA final concentration was 5nM, 0.5nM or 0.05nM. After transfection for 48h, the cell culture solution in each well was discarded, and 50uL of cell lysis solution (Jimma Gene) was added to each well. After incubation for 5min, 5uL of stop solution was added to each well to obtain the final lysis product.

[0358] Mock group: no siRNA was added, and DEPC water was added.

[0359] Step three: qPCR detection of the knockdown effect of modified hLPA siRNA on target gene mRNA

[0360] (1) RT-qPCR detection

[0361] The RNA template was obtained after the cell lysate was treated by adding DNase I to remove genomic DNA. The RT-qPCR probe method reaction system shown in Table 7 was prepared, and the above RNA was subjected to a fluorescent quantitative PCR reaction on an LC480 through the reaction system shown in Table 7, the primers shown in Table 6, and the program shown in Table 8.

[0362] Table 6. hLPA and Hgapdh qPCR primer sequence information

[0363] (2) RT-qPCR probe method reaction system

[0364] Table 7, RT-qPCR probe method reaction system

[0365] (3) On-machine detection

[0366] The fluorescent quantitative PCR reaction was performed on an LC480, and the program was as follows:

[0367] Table 8, RT-qPCR reaction program

[0368] The comparative Ct (ΔΔCt) method was used to perform relative quantitative calculation on the target gene LPA in each test group, and the calculation method was as follows:

[0369] ΔCt (test group) = Ct (test group target gene) - Ct (test group internal reference gene)

[0370] ΔCt (control group) = Ct (control group target gene) - Ct (control group internal reference gene)

[0371] ΔΔCt (test group) = ΔCt (test group) - ΔCt (control group average)

[0372] ΔΔCt (control group) = ΔCt (control group) - ΔCt (control group average)

[0373] Among them, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) of each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔΔCt value.

[0374] Taking the control group as the reference, the expression level of LPA mRNA in the test group was normalized, and the expression level of LPA mRNA in the control group was defined as 100%.

[0375] Test group LPA mRNA relative expression level = 2 ^-ΔΔCt (test group) x 100%

[0376] In the above formula, the control group is the Mock group.

[0377] The detection results are shown in Table 9.

[0378] Table 9 Verification results of siRNA sequences for inhibiting hLPA expression

[0379] Example 4 In vitro experiment of modified siRNA for inhibiting hLPA expression

[0380] Different siRNAs modified by M3 and 5'(E)-VP were subjected to in vitro cell screening. In this example, 293T / hLPA stable transfection cells in Example 3 were used. Transfection reagent Lipofectamine RNAiMAX was used for siRNA transfection. According to the method described in the instructions, the transfection complex was prepared. For example, for the preparation of single-well transfection complex, siRNA was diluted with opti-MEM to prepare siRNA diluent, and 25 μL of siRNA diluent was taken per well; 25 μL of opti-MEM was mixed with 0.25 μL of Lipofectamine RNAiMAX transfection reagent to prepare transfection reagent diluent. The above two diluents were mixed and gently mixed, and then incubated at room temperature for 10-20 minutes, and then added to the corresponding well of the cell plate. The final concentration of siRNA was 5 nM, 0.5 nM or 0.05 nM. After transfection for 48 h, the cell culture solution in each well was discarded, 50 μL of cell lysis solution (Gimaa Gene) was added to each well, and after incubation for 5 min, 5 μL of stop solution was added to obtain the final lysis product.

[0381] Mock group: no siRNA was added, and DEPC water was added.

[0382] qPCR detection of the knockdown effect of modified hLPA siRNA on target gene mRNA and data analysis method, refer to the experimental steps described in Example 3.

[0383] The inhibitory effect of different modified sequences on hLPA mRNA expression is shown in Table 10.

[0384] Table 10 Verification results of siRNA modified sequences for inhibiting hLPA expression

[0385] Example 5 In vivo experiment of modified siRNA in hLPA transgenic mice

[0386] The inhibitory effect of M1 modified sequence on hLPA mRNA expression was detected in pAlb-hLPA-Tg humanized mice.

[0387] In the experiment, pAlb-hLPA-Tg humanized mice (female) in C57BL / 6J background were randomly grouped by body weight, and were respectively treated with sense strand 3' end L96 coupled M1 modified siRNA or normal saline. The siRNA was injected subcutaneously at a dose of 1 mg / kg, and the injection volume of each mouse was 200 μL. The liver samples of the mice were taken on the day before administration (Day-1) and the 21st day after administration, and after reverse transcription, the mRNA expression of LPA was detected by qPCR. The experimental operation and data analysis of qPCR are described in Example 3. The results are shown in Table 11.

[0388] Table 11 In vivo inhibition effect of M1 modified sequence on LPA mRNA

[0389] Example 6, in vivo activity detection of modified sequence

[0390] C57BL / 6J background pAlb-hLPA-Tg humanized female mice were randomly grouped by body weight, and were respectively treated with L96 coupled modified sequence (see Table 3 for sequence information), normal saline. L96 coupled modified siRNA was injected subcutaneously at a dose of 1 mg / kg, and the injection volume of each mouse was 200 μL. The serum samples of the mice were taken on the day before administration (Day-1) and the 7th day, 14th day, 21st day, and 28th day after administration. The human Apo(a) protein level was detected using an ELISA kit specific for human LPA (Mercodia; CAT#10-1106-01) according to the manufacturer's instructions. The standardization method of Apo(a) protein level is as follows: at each time point, the Apo(a) protein level of each mouse is divided by its expression level before administration (Day-1) to obtain the "normalized to pre-treatment" expression ratio; then, the "normalized to pre-treatment" expression ratio at a specific time point is divided by the average "normalized to pre-treatment" expression ratio of all mice in the saline control group at that time point, so as to standardize the expression level at each time point to the saline control group. The results are shown in Table 12.

[0391] Table 12 In vivo inhibition effect of different modified sequences on Apo(a) expression in hLPA transgenic mice

[0392] wherein olpasiran is shown in FIG. 1 of CN 118019566 A.

[0393] Example 8, in vivo experiment of modified siRNA in hLPA transgenic mice

[0394] C57BL / 6J background pAlb-hLPA-Tg humanized mice were randomly grouped by body weight, 3 female mice in each group, and were treated with L96 conjugated siRNA Lpa-35M3GVP, Lpa-176M3GVP, Lpa-276M3GVP, Lpa-373M3GVP, Lpa-395M3GVP, Lpa-84M3GVP, Lpa-44M3GVP, Lpa-476M3GVP, Lpa-482M3GVP, Lpa-511M3GVP, respectively, which were conjugated with (E)-VP at the 5' end of the antisense strand and modified by M3, and normal saline treatment. Among them, L96 conjugated siRNA was injected subcutaneously at a dose of 1 mg / kg, and the volume of each mouse was 200 μL. The serum samples of mice were taken on the day before administration (Day-1) and the 7th day, 14th day, 21st day, 28th day, 35th day and 42nd day, 49th day, 56th day after administration. The human Apo(a) protein level was detected using an ELISA kit specific for human LPA (Mercodia; CAT#10-1106-01) according to the manufacturer's instructions. The standardization method of Apo(a) protein level, at each time point, the Apo(a) protein level of each mouse was divided by its expression level before administration (Day-1) to obtain the "normalized to pre-treatment" expression ratio; then, the "normalized to pre-treatment" expression ratio at a specific time point was divided by the average "normalized to pre-treatment" expression ratio of all mice in the normal saline control group at that time point, so that the expression level at each time point was standardized to the normal saline control group. The results are shown in Table 13 below.

Claims

1. A double-stranded RNA molecule, wherein the double-stranded RNA molecule comprises a sense strand and an antisense strand forming at least in part a double-stranded region, the antisense strand comprising 17-21 consecutive nucleotides of the nucleotide sequence of any even-numbered one of SEQ ID NO. 1-548, the sense strand comprising 17-19 consecutive nucleotides of the nucleotide sequence of any odd-numbered one of SEQ ID NO. 1-548.

2. The double-stranded RNA molecule according to claim 1, wherein the length of the sense strand is not more than 30 nucleotides, and / or the length of the antisense strand is not more than 30 nucleotides, preferably the length of the sense strand is not more than 21 nucleotides, and / or the length of the antisense strand is not more than 23 nucleotides, more preferably the length of the sense strand is not more than 19 nucleotides, and / or the length of the antisense strand is not more than 21 nucleotides.

3. The double-stranded RNA molecule according to claim 1 or 2, wherein the sense strand and the antisense strand of the double-stranded RNA molecule comprise or are any combination selected from: the sense strand of nucleotide sequence number n and the antisense strand of nucleotide sequence number n+1, wherein n is an odd number from 1-548.

4. A modified double-stranded RNA molecule comprising any one of claims 1-3, wherein at least one nucleotide is chemically modified, preferably, the chemical modification comprising replacing the 2' hydroxyl group of the ribosyl group of the nucleotide with another group, and / or modifying the bases on the nucleotide, and / or replacing the 5' hydroxyl group of the ribosyl group of the nucleotide with another group, and / or modifying the phosphate ester, more preferably, the chemically modified nucleotide is selected from at least one of the following: 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, 5'-(E)-vinylphosphonate modified nucleotides, 3'-thiophosphate modified nucleotides, 2'-methyl modified nucleotides, deoxy-nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides (such as LNA), non-locked nucleotides, configuration-restricted nucleotides. The chemically modified nucleotides include: restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, glycerol nucleotides (GNA), open-ring nucleotides (UNA), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate groups, and nucleotides containing 5'-phosphate mimics. More preferably, the chemically modified nucleotides are selected from at least one of the following: locked nucleotides (such as LNA), 2'-deoxy-nucleotides, 2'-methoxy-modified nucleotides, 2'-fluoro-modified nucleotides, 5'-(E)-vinylphosphonate-modified nucleotides, and 3'-thiophosphate-modified nucleotides.

5. The modified double-stranded RNA molecule according to claim 4, optionally with an invab attached to the 5' and / or 3' end of the sense strand.

6. The modified double-stranded RNA molecule according to claim 4 or 5, wherein the double-stranded RNA molecule is selected from any one of the following: (1) The justice chain is: UmsUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO.549), The ansense chain is: AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO.550); (2) The chain of justice is: (invab)sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms(invab) (SEQ ID NO. 551), AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550); (3) sense strand is: (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552), AmsGfsAmsUmGmAmCmCmAmAmGmCfUmUfGmGfCmAmAmGmsUm (SEQ ID NO. 553); (4) sense strand is: (invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 551), AmsGfsAmUmGmAfCmCmAmAmGmCfUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 554); (5) sense strand is: UmsUmsGmCmCmAmAfGfC(d)UmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 555), AmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 550); (6) sense strand is: T(LNA) sUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 556), AmsGfsAmUmGmAfCmCfAfAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 557); (7) sense strand is: UmsAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 558), UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559); (8) sense strand is: T(LNA) sAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 560), UmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 561); (9) sense strand is: (invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms (invab) (SEQ ID NO. 562), the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559); (10) the sense strand is: (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552), the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559); (11) the sense strand is: (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552), the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559); (12) the sense strand is: UmsAmsUmCmGmAmGfGfC(d)AmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 566), the antisense strand is: UmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 559); (13) the sense strand is: UmsUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 549), the antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567); (14) the sense strand is: (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552), the antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567); (15) the sense strand is: (invab) sAmCmUmUmGmCmCfAmAfGmCfUmUmGmGmUmCmAmUmCmUms (invab) (SEQ ID NO. 552), the antisense strand is: VPAmsGfsAmsUmGmAmCmCmAmAmGmCfUmUfGmGfCmAmAmGmsUm (SEQ ID NO. 568); (16) the sense strand is: (Invab) sAmCmUmUmGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUms (Invab) (SEQ ID NO. 551), the antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCfUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 569); (17) the sense strand is: UmsUmsGmCmCmAmAfGfC(d)UmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 555), the antisense strand is: VPAmsGfsAmUmGmAfCmCmAmAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 567); (18) the sense strand is: T(LNA) sUmsGmCmCfAmAfGfCfUmUmGmGmUmCmAmUmCmUm (SEQ ID NO. 556), the antisense strand is: VPAmsGfsAmUmGmAfCmCfAfAmGmCmUmUfGmGfCmAmAmsGmsUm (SEQ ID NO. 570); (19) the sense strand is: UmsAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 558), the antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 571); (20) the sense strand is: T(LNA) sAmsUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 560), the antisense strand is: VPUmsGfsGmAmGmAfAmUfGfUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 572); (21) the sense strand is: (invab) sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms (invab) (SEQ ID NO. 562), the antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 571); (22) the sense strand is: (invab) sGmUmUmAmUmCmGfAmGfGmCfAmCmAmUmUmCmUmCmCmAms (invab) (SEQ ID NO. 563), the antisense strand is: VPUmsGfsGmsAmGmAmAmUmGmUmGmCfCmUfCmGfAmUmAmAmsCm (SEQ ID NO. 573); (23) the sense strand is: (Invab)sGmUmUmAmUmCmGfAmGfGfCfAmCmAmUmUmCmUmCmCmAms(Invab) (SEQ ID NO. 562), the antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCfCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 574); (24) the sense strand is: UmsAmsUmCmGmAmGfGfC(d)AmCmAmUmUmCmUmCmCmAm (SEQ ID NO. 566), the antisense strand is: VPUmsGfsGmAmGmAfAmUmGmUmGmCmCmUfCmGfAmUmAmsAmsCm (SEQ ID NO. 571); wherein Af represents 2'-fluoro-adenosine-3'-phosphate, Afs represents 2'-fluoro-adenosine-3'- phosphorothioate, Am represents 2'-methoxy-adenosine-3'-phosphate, Ams represents 2'- methoxy-adenosine-3'-phosphorothioate, Cf represents 2'-fluoro-cytidine-3'-phosphate, Cfs represents 2'-fluoro-cytidine-3'-phosphorothioate, Cm represents 2'-methoxy-cytidine-3'- phosphate, Cms represents 2'-methoxy-cytidine-3'-phosphorothioate, Gf represents 2'-fluoro- guanosine-3'-phosphate, Gfs represents 2'-fluoro-guanosine-3'-phosphorothioate, Gm represents 2'-methoxy-guanosine-3'-phosphate, Gms represents 2'-methoxy-guanosine-3'- phosphorothioate, Uf represents 2'-fluoro-uridine-3'-phosphate, Ufs represents 2'-fluoro- uridine-3'-phosphorothioate, Um represents 2'-methoxy-uridine-3'-phosphate, Ums represents 2'-methoxy-uridine-3'-phosphorothioate, T(LNA)s represents C(d) represents 2' deoxy-cytidine-3' phosphate 5' end (invab) s means: 3' end (invab) means: VP indicates the hydroxyl group at the 5' position of the nucleotide is replaced by (E)-vinyl phosphate.

7. The modified double stranded RNA molecule of any one of claims 4-6, wherein the 3'-end of the sense strand is coupled to a ligand comprising N-acetylgalactosamine (GalNAc) or a derivative thereof, preferably the ligand is one or more GalNAc derivatives attached via a monovalent, divalent, trivalent or tetravalent branched linker.

8. The modified double stranded RNA molecule of claim 7, wherein the ligand comprises a compound of the formula: ###0001### the compound is coupled to the sense strand via a phosphonate or thiophosphonate.

9. A dimeric siRNA comprising the double stranded RNA molecule of any one of claims 1-3 or the modified double stranded RNA molecule of any one of claims 4-8.

10. A pharmaceutical composition comprising the double stranded RNA molecule of any one of claims 1-3 or the modified double stranded RNA molecule of any one of claims 4-8 or the dimeric siRNA of claim 9, and a pharmaceutically acceptable carrier.

11. Use of the double stranded RNA molecule of any one of claims 1-3 or the modified double stranded RNA molecule of any one of claims 4-8 or the dimeric siRNA of claim 9 or the pharmaceutical composition of claim 10 in any one of: D1) the manufacture of a composition for inhibiting expression of an LPA gene; D2) the inhibition of expression of an LPA gene; D3) the treatment of a disease associated with an LPA gene target; D4) the manufacture of a composition for the treatment of a disease associated with an LPA gene target.

12. A method of treating a disease associated with an LPA gene target, comprising administering to a subject a therapeutically effective amount of the double stranded RNA molecule of any one of claims 1-3 or the modified double stranded RNA molecule of any one of claims 4-8 or the dimeric siRNA of claim 9 or the pharmaceutical composition of claim 10.

13. A method of inhibiting expression of an LPA gene in a cell, the method comprising: contacting the cell with the double-stranded RNA molecule of any one of claims 1-3, or the modified double-stranded RNA molecule of any one of claims 4-8, or the dimeric siRNA of claim 9, or the pharmaceutical composition of claim 10; maintaining the cell for a time sufficient to achieve degradation of the mRNA transcript of the LPA gene to inhibit expression of the LPA gene in the cell; Preferably, the cell is in a subject in vivo or in vitro. More preferably, the subject is suffering from a disease associated with the LP(A) gene target.

14. Use according to claim 11, or method according to claim 12 or 13, wherein the disease associated with the LP(A) gene target is selected from one or more of the group consisting of: Berger's disease, peripheral arterial disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, ischemic stroke, heart failure, atrial fibrillation, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, venous thrombosis.

15. Use of the double-stranded RNA molecule of any one of claims 1-3, or the modified double-stranded RNA molecule of any one of claims 4-8, for the preparation of a dimeric siRNA.

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