Dsrna molecule for regulating LPA expression

Abstract

Provided are a double-stranded RNA (dsRNA) for inhibiting the expression of an LPA gene, a vector and a cell comprising nucleotides encoding same, and a method for using the dsRNA, the vector or the cell to treat apolipoprotein (a)-mediated or related diseases or symptoms in a subject.

Description

dsRNA molecules modulating expression of LPA

[0001] This application claims priority to Chinese Patent Application No. 202410733556.5, filed on June 6, 2024, entitled “dsRNA molecules modulating expression of LPA,” the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present invention relates to the field of RNA interference. BACKGROUND

[0003] Lipoproteins are globular particles composed of a nonpolar core of acylglycerols and cholesteryl esters and a surrounding amphipathic coat of proteins, phospholipids, and cholesterol. Based on their function and physical properties, lipoproteins are classified into five major groups: chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL).

[0004] Lipoprotein(a) (also known as Lp(a)) is a pro-atherogenic LDL-like particle that is rich in cholesterol. Lipoprotein(a) exhibits inter-individual differences in concentration that range from less than 0.1 mg / dL to greater than 300 mg / dL, hundreds of fold. Plasma Lp(a) concentrations above 30 mg / dL, and even above 50 mg / dL, are associated with increased risk of cardiovascular disease, including coronary artery disease (ASCVD), myocardial infarction, stroke, aortic valve stenosis, heart failure, peripheral artery disease, and all-cause mortality. The 2016 Chinese Guidelines for Prevention and Treatment of Dyslipidemia in Adults defined >30 mg / dL as Lp(a) abnormality. Using this as a criterion, approximately 30% of patients with prior cardiovascular events in China have Lp(a) abnormality.

[0005] Lipoprotein(a) contains two different proteins linked by disulfide bonds, apolipoprotein(a) (or ApoA) and apolipoprotein B (or ApoB). Apolipoprotein(a) is a unique apolipoprotein encoded by the LPA gene that has been shown to specifically control the physiological concentration of Lp(a). Its size difference is due to differences in the number of 5.5-kb sequences encoding kringle-4 in the LPA gene that are tandemly repeated between alleles. The LPA gene is primarily expressed by the liver, and is almost exclusively synthesized in the liver.

[0006] Increased expression of apolipoprotein(a) causes elevated plasma lipoprotein(a) levels, leading to increased risk of atherosclerosis and its manifestations, including hypercholesterolemia.

[0007] Plasma Lp(a) concentration is strongly influenced by genetic factors and is refractory to most drugs.

[0008] Some therapies that are primarily aimed at lowering low-density lipoprotein cholesterol also have an effect on Lp(a) concentration. These are primarily PCSK9 inhibitors, which can lower LDL cholesterol by 60% and Lp(a) by 25-30%. In addition, lipoprotein apheresis can lower Lp(a) and LDL cholesterol by about 60-70%. However, there are currently no drugs that specifically lower apolipoprotein(a) in the clinic.

[0009] There is a need in the art for compositions and methods for treating diseases, disorders, and conditions associated with apolipoprotein(a).

[0010] Reducing LPA expression through double-stranded RNA (dsRNA), in particular small interfering RNA (siRNA), based on the RNA interference mechanism, is a new approach to treating diseases, disorders, and conditions associated with lipoprotein a. SUMMARY

[0011] The present invention provides novel double-stranded RNAs (or simply dsRNAs), vectors, cells, and pharmaceutical compositions and kits comprising the same, and methods of using the same to inhibit or reduce LPA gene expression or to treat diseases or conditions that benefit from a reduction in LPA gene expression.

[0012] In a first aspect, the present invention provides a double-stranded RNA (dsRNA) for inhibiting LPA gene expression, the dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82. In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41.

[0013] In some embodiments, a hairpin loop is formed between the sense strand and the antisense strand in the dsRNA. In other embodiments, the dsRNA is an siRNA.

[0014] In some embodiments, the sense strand and the antisense strand are each independently 15-27 nucleotides in length, preferably 18-25 nucleotides in length, more preferably 19-21 nucleotides in length. In some embodiments, the sense strand is 15-27 nucleotides in length, preferably 17-25 nucleotides in length, more preferably 18-23 nucleotides in length, more preferably 19-21 nucleotides in length, most preferably 19 nucleotides in length. In some embodiments, the antisense strand is 15-27 nucleotides in length, preferably 17-25 nucleotides in length, more preferably 18-23 nucleotides in length, more preferably 19-22 nucleotides in length, most preferably 21 nucleotides in length.

[0015] In some embodiments, the double-stranded region is 15-25 nucleotide pairs in length, preferably 16-23 nucleotide pairs in length, more preferably 18-20 nucleotide pairs in length, most preferably 19 nucleotide pairs in length.

[0016] In some embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 1 nucleotide, for example one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 2 nucleotides. In some embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least 1 nucleotide, preferably the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides. In some specific embodiments, the dsRNA has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0017] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, a nucleotide sequence of at least 18 contiguous nucleotides, a nucleotide sequence of at least 19 contiguous nucleotides, or a nucleotide sequence of at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82, preferably the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82.

[0018] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, or a nucleotide sequence of at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41, preferably the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41.

[0019] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides, a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, a nucleotide sequence of at least 18 contiguous nucleotides, a nucleotide sequence of at least 19 contiguous nucleotides, or a nucleotide sequence of at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 48, 50, 55, 68, 80, 81, and 82. In some preferred embodiments, the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 48, 50, 55, 68, 80, 81, and 82.

[0020] In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides, a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, or a nucleotide sequence of at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 14, 27, 39, 40, and 41. In some preferred embodiments, the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 14, 27, 39, 40, and 41.

[0021] In some embodiments, the dsRNA comprises any one of the pairs of paired sense strand sequence and antisense strand sequence as shown in Table 3 of the specification. In some embodiments, in the dsRNAs of the present application for inhibiting expression of LPA in a cell,

[0022] (1) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 3, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 44;

[0023] (2) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 7, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 48;

[0024] (3) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 9, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 50;

[0025] (4) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 14, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 55;

[0026] (5) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 27, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 68;

[0027] (6) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 39, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 80;

[0028] (7) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 40, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 81; or

[0029] (8) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 41, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 82.

[0030] In some embodiments, substantially all of the nucleotides of the sense strand and / or substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and / or all of the nucleotides of the antisense strand are modified nucleotides.

[0031] In some embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of: 2'-0-alkyl modified nucleotides (e.g., 2'-0-methyl modified nucleotides), 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides comprising phosphorothioate groups, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-0-allyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases, terminal nucleotides linked to a cholesteryl derivative or dodecanoic acid didecanoyl amide group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, glycol modified nucleotides (GNAs), and 2-0-(N-methylacetamide) modified nucleotides. In some embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, and nucleotides comprising phosphorothioate groups.

[0032] In some embodiments, the sense strand and / or the antisense strand comprises at least 2 2'-fluoro-modified nucleotides. In some embodiments, the sense strand and / or the antisense strand comprises at least 8 2'-0-methyl-modified nucleotides. In some embodiments, the 3' and / or 5' terminus of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate internucleotide linkages, preferably 2-3 phosphorothioate internucleotide linkages.

[0033] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0034] (i) (counting from the 5' end) 2'-0-methyl-modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro-modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and / or

[0035] (ii) (counting from the 5' end) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21.

[0036] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0037] (i) (counting from the 5' end) 2'-0-methyl-modified nucleotides at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21;

[0038] (ii) (counting from the 5' end) 2'-fluoro-modified nucleotides at positions 2, 7, 12, 14, and 16;

[0039] (iii) (counting from the 5' end) a SCP-modified nucleotide at position 1; and / or

[0040] (iv) (counting from the 5' end) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21.

[0041] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0042] (i) (counting from the 5' end) 2'-0-methyl-modified nucleotides at positions 1, 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21;

[0043] (ii) 2'-fluoro modified nucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end;

[0044] (iii) an SCP modified nucleotide at position 1, counting from the 5' end; and / or

[0045] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21, counting from the 5' end.

[0046] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0047] (i) 2'-O-methyl modified nucleotides at positions 1, 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21, counting from the 5' end;

[0048] (ii) 2'-fluoro modified nucleotides at position 14, counting from the 5' end;

[0049] (ii) 2'-deoxy modified nucleotides at positions 2, 5, 7, and 12, counting from the 5' end;

[0050] (iv) an SCP modified nucleotide at position 1, counting from the 5' end; and / or

[0051] (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21, counting from the 5' end.

[0052] In some embodiments, the sense strand of the dsRNA has a length of 19 nucleotides and has:

[0053] (i) 2'-O-methyl modified nucleotides at positions 1-6, 10-19, and 2'-fluoro modified nucleotides at positions 7-9, counting from the 5' end; and / or

[0054] (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5' end.

[0055] In some embodiments, the antisense strand of the dsRNA comprises any one of the nucleotide sequences set forth in Table 5, and / or the sense strand comprises any one of the nucleotide sequences set forth in Table 4A.

[0056] In some embodiments, the dsRNA comprises any of the paired sense strand sequence and antisense strand sequence set forth in Table 6A.

[0057] In some preferred embodiments, the dsRNA of the application comprises a sense strand and an antisense strand, wherein

[0058] (1) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 121), and the antisense strand comprises (SCP-U) sAfsUmAmAmCmUfCmUmGmUmCfCmAfUmUfAmCmCmsGmsUm (SEQ ID NO: 342);

[0059] (2) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 122), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsGmsUm (SEQ ID NO: 305);

[0060] (3) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAm (SEQ ID NO: 123), and the antisense strand comprises (SCP-U) sdAsUmCmdAAmdGCmCmAmGmdCAmUfUmUmGmGmGmsUmsAm (SEQ ID NO: 317);

[0061] (4) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAm (SEQ ID NO: 124), and the antisense strand comprises (SCP-U) sAfsUmCmAmAfGmCmCmAmGmCfAmUfUmUfGmGmGmsUmsAm (SEQ ID NO: 309);

[0062] (5) the sense strand comprises UmsGmsGmUmAmAmUfGfGfAmCmAmGmAmGmUmUmAmsAm (SEQ ID NO: 125), and the antisense strand comprises (SCP-U) sUfsAmAmCmUmCfUmGmUmCmCfAmUfUmAfCmCmAmsUmsGm (SEQ ID NO: 340);

[0063] (6) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAm (SEQ ID NO: 126), and the antisense strand comprises (SCP-U)sdAsGmAmdTGmdACmCmAmAmdGCmUfUmGmGmCmAmsAmsGm (SEQ ID NO: 334);

[0064] (7) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAm (SEQ ID NO: 127), and the antisense strand comprises (SCP-U)sAfsGmAmUmGmAfCmCmAmAmGfCmUfUmGfGmCmAmsAmsGm (SEQ ID NO: 314);

[0065] (8) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 128), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsCmsUm (SEQ ID NO: 345);

[0066] (9) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 129), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsAmsUm (SEQ ID NO: 346); or

[0067] (10) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 130), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsUmsUm (SEQ ID NO: 347).

[0068] In some embodiments, the dsRNA is further conjugated to a ligand moiety.

[0069] The ligand moiety preferably comprises N-acetylgalactosamine, preferably the sense strand of the dsRNA is conjugated to the ligand moiety. In some embodiments, the 3’ end of the sense strand is conjugated to the ligand moiety. In other embodiments, the 5’ end of the sense strand is conjugated to the ligand moiety.

[0070] In some embodiments, the ligand moiety comprises a conjugate group represented by Formula (X’):

[0071] wherein,

[0072] represents the position of attachment to the dsRNA;

[0073] Q is independently H,

[0074] wherein L1is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0075] L2is a bond or -CH2CH2C(O)-;

[0076] L3is a bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0077] L4is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -;

[0078] wherein a = 0, 1, 2, or 3;

[0079] b = 1, 2, 3, 4, or 5;

[0080] c = 1, 2, 3, 4, or 5;

[0081] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0082] L is a bond, -CH2O-, or -NHC(O)-;

[0083] L’ is a bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O) e -;

[0084] wherein e is 1, 2, 3, 4, or 5;

[0085] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0086] wherein M is

[0087] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0088] or R1and R3together form -C 1-2 alkylene-, and R2is H;

[0089] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0090] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0091] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0092] In some embodiments, the conjugated ligand targets the asialoglycoprotein receptor (ASGPR).

[0093] In some preferred embodiments, the conjugate group is selected from Table 1:

[0094] Table 1. Structures of conjugate groups

[0095] In some preferred embodiments, the conjugate group is selected from Table 2:

[0096] Table 2. Structures of conjugate groups

[0097] In some embodiments, the ligand moiety in the dsRNA has the following structure:

[0098] wherein denotes the position of attachment to the dsRNA, the sense strand of the dsRNA, via a phosphonate group or a thiophosphonate group.

[0099] In some embodiments, the ligand moiety in the dsRNA has the following structure:

[0100] wherein indicates the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, through a phosphonate or thiophosphonate group.

[0101] In some embodiments, the ligand moiety in the dsRNA of the application has the following structure:

[0102] wherein indicates the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, through a phosphonate or thiophosphonate group.

[0103] In some embodiments, the ligand moiety in the dsRNA of the application has the following structure:

[0104] wherein indicates the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, through a phosphonate or thiophosphonate group.

[0105] In some embodiments, the sense strand of the dsRNA comprises any one of the nucleotide sequences set forth in Table 4B of the specification.

[0106] In some embodiments, the dsRNA comprises any one pair of paired sense strand sequence and antisense strand sequence set forth in Table 6B of the specification.

[0107] In some preferred embodiments, the dsRNA comprises any one pair of paired sense strand sequence and antisense strand sequence:

[0108] (1) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U)sAfsUmAmAmCmUfCmUmGmUmCfCmAfUmUfAmCmCmsGmsUm (SEQ ID NO: 342);

[0109] (2) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsGmsUm (SEQ ID NO: 305);

[0110] (3) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAms-GL6 (SEQ ID NO: 182), and the antisense strand comprises (SCP-U)sdAsUmCmdAAmdGCmCmAmGmdCAmUfUmUmGmGmGmsUmsAm (SEQ ID NO: 317);

[0111] (4) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAms-GL6 (SEQ ID NO: 182), and the antisense strand comprises (SCP-U)sAfsUmCmAmAfGmCmCmAmGmCfAmUfUmUfGmGmGmsUmsAm (SEQ ID NO: 309);

[0112] (5) the sense strand comprises UmsGmsGmUmAmAmUfGfGfAmCmAmGmAmGmUmUmAmsAms-GL6 (SEQ ID NO: 184), and the antisense strand comprises (SCP-U)sUfsAmAmCmUmCfUmGmUmCmCfAmUfUmAfCmCmAmsUmsGm (SEQ ID NO: 340);

[0113] (6) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAms-GL6 (SEQ ID NO: 200), and the antisense strand comprises (SCP-U)sdAsGmAmdTGmdACmCmAmAmdGCmUfUmGmGmCmAmsAmsGm (SEQ ID NO: 334);

[0114] (7) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAms-GL6 (SEQ ID NO: 200), and the antisense strand comprises (SCP-U)sAfsGmAmUmGmAfCmCmAmAmGfCmUfUmGfGmCmAmsAmsGm (SEQ ID NO: 314);

[0115] (8) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsCmsUm (SEQ ID NO: 345);

[0116] (9) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsAmsUm (SEQ ID NO: 346); or

[0117] (10) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsUmsUm (SEQ ID NO: 347).

[0118] In a second aspect, the present application provides a cell containing a dsRNA or a vector of the present application.

[0119] In a third aspect, the present application provides a pharmaceutical composition comprising a dsRNA or a cell of the present application, and optionally a pharmaceutically acceptable carrier or excipient.

[0120] In a fourth aspect, the present application provides a kit comprising a dsRNA, a cell, or a pharmaceutical composition of the present application.

[0121] In a fifth aspect, the present application provides a method of reducing apolipoprotein (a) in a subject, the method comprising the step of administering to the subject a dsRNA, a cell, or a pharmaceutical composition of the present application. The present application also provides a method of treating a disease or disorder in a subject that benefits from a reduction in LPA gene expression, the method comprising the step of administering to the subject a dsRNA, a cell, or a pharmaceutical composition of the present application. The present application also provides a method of preventing at least one symptom in a subject having a disease or disorder that benefits from a reduction in LPA gene expression, the method comprising the step of administering to the subject a dsRNA, a cell, or a pharmaceutical composition of the present application.

[0122] In some implementations, the disease or condition that benefits from reduced LPA gene expression is an apolipoprotein(a)-mediated disease or an apolipoprotein(a)-associated disease.

[0123] In some embodiments, the apolipoprotein(a)-mediated disease or apolipoprotein(a)-related disease is selected from the group consisting of: Bergey's disease, peripheral artery 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, hyperlipoproteinemia, atherosclerosis (e.g., cerebrovascular atherosclerosis), cerebrovascular disease, venous thrombosis, myocardial infarction, dyslipidemia, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis. In some embodiments, the method of the present invention for treating a subject with a disease or condition that benefits from reduced LPA gene expression, for preventing at least one symptom in a patient with a disease or condition that benefits from reduced LPA gene expression, or for reducing apolipoprotein (a) in a subject comprises administering the dsRNA, cells, or pharmaceutical composition subcutaneously, locally, or intravenously to the subject. In some embodiments, the subject is a human patient.

[0124] Invention Details

[0125] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0126] It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0127] In this specification and claims, unless otherwise expressly stated herein, the singular forms “a,” “one,” and “this” include the plural forms.

[0128] When the embodiments give numerical ranges, it is understood that, unless the present invention indicates otherwise, each numerical range's two endpoints, and any number in between the two endpoints, can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. Other features, objects, and advantages of the invention will be apparent from the description of the preferred embodiments that follow.

[0129] Definitions

[0130] A "double-stranded region" refers herein to a region comprising two nucleic acid strands that are antiparallel and complementary or substantially complementary.

[0131] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a ribonucleic acid molecule or a complex of ribonucleic acid molecules comprising a double-stranded region as defined above. The two parts forming the double-stranded region can be two different parts of one larger RNA molecule, or they are separate RNA molecules.

[0132] When the two parts are separate RNA molecules, the dsRNA herein is referred to as a small interfering RNA or short interfering RNA, siRNA for short.

[0133] When the two parts are two different parts of one larger molecule, i.e. when the 3' end of one part is linked to the 5' end of the other part by one or more uninterrupted nucleotides between the two, the uninterrupted nucleotides used for the linkage are referred to as "hairpin loop". When the two parts are covalently linked to form the double-stranded region by other means than a hairpin loop, the linkage structure is referred to as "linker". Such dsRNA is cleaved into siRNA by an endoribonuclease in the cell, referred to as Dicer enzyme, after introduction into the cell.

[0134] The term "siRNA" herein is a class of double-stranded RNA molecules comprising a sense strand and an antisense strand that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary or substantially complementary to the antisense strand. The siRNA is typically double-stranded, comprising an antisense strand that is complementary to a target RNA, and a sense strand that is complementary or substantially 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. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0135] As used herein, the term "antisense strand" refers to a strand in a dsRNA (particularly siRNA) that comprises a region that is fully or substantially complementary to a target sequence.

[0136] As used herein, the term "complementary region" refers to a region on the antisense strand that is fully or substantially complementary to a target mRNA sequence. Where the complementary region is not fully complementary to the target sequence, the mismatches can be in the interior or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' and / or 3' end. The portion of the antisense strand that is most sensitive to mismatches is referred to as the "seed region." For example, in a 19 nt siRNA, the 19th position (from 5' to 3') can tolerate some mismatches.

[0137] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, e.g., stringent conditions. For example, stringent conditions can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50°C or 70°C for 12-16 hours.

[0138] As used herein, "complementary" sequences can also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, insofar as they meet the above requirements with respect to their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0139] As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding apolipoprotein(a)). For example, a polynucleotide is at least partially complementary to an mRNA encoding apolipoprotein(a) if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding apolipoprotein(a).

[0140] The terms "complementary," "fully complementary," and "substantially complementary" as used herein can be used in reference to base pairing between the sense strand and the antisense strand of a dsRNA, particularly an siRNA, or between the antisense strand of a dsRNA, particularly an siRNA, and a target sequence.

[0141] The term "sense strand" as used herein refers to the strand of an siRNA that includes a region that is substantially complementary to a region that is an antisense strand as defined herein.

[0142] A "nucleoside" is a compound consisting of a purine or pyrimidine base, and either ribose or deoxyribose, a "nucleotide" is a compound consisting of a purine or pyrimidine base, ribose or deoxyribose, and a phosphate, and an "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) of, for example, less than 100, 200, 300, or 400 nucleotides in length.

[0143] A "base" is the basic building block of synthetic nucleosides, nucleotides, and nucleic acids, which contains nitrogen in its constituent elements, also known as a "nitrogenous base." Herein, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of a nucleotide, which are adenine, uracil, thymine, guanine, and cytosine, respectively.

[0144] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that overhangs from the double-stranded region of an siRNA. A nucleotide overhang is present, for example, 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, an overhang of at least two nucleotides, an overhang of at least three nucleotides, an overhang of at least four nucleotides, an overhang of at least five nucleotides or more. The nucleotide overhang can comprise or consist of nucleotides / modified nucleotides (including deoxynucleotides / nucleosides). The overhang(s) can be on the sense strand or the antisense strand or any combination thereof. An overhang of one or more nucleotides can be present at the 5 '-end, the 3 '-end, or both ends of the antisense or sense strand of an siRNA.

[0145] A "blunt end" or "blunt end" or "blunt end" means that there are no unpaired nucleotides at the end of the double-stranded siRNA, i.e., no nucleotide overhang. A "blunt-end siRNA" is an siRNA that is double-stranded over the entire length of the siRNA, i.e., there is no nucleotide overhang at either end of the molecule.

[0146] The dsRNA (especially siRNA) of the present invention comprises substantially all modified nucleotides. For example, substantially all nucleotides of the sense strand are modified nucleotides, or substantially all nucleotides of the antisense strand are modified nucleotides, or substantially all nucleotides of both the sense and antisense strands are modified nucleotides. In other embodiments of the invention, all nucleotides of the dsRNA (especially siRNA) of the present invention are modified nucleotides. For example, substantially all nucleotides of the sense strand are modified nucleotides, or substantially all nucleotides of the antisense strand are modified nucleotides, or substantially all nucleotides of both the sense and antisense strands are modified nucleotides. As used herein, “substantially all nucleotides are modified” means that the majority, but not all, nucleotides of the dsRNA (especially siRNA) of the present invention are modified, and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0147] In this document, "modified nucleotides" include, but are not limited to, 2'-O-alkyl-modified nucleotides (e.g., 2'-O-methyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abase-free nucleotides, reverse abase-free deoxyribonucleotides, nucleotides containing a thiophosphate group, vinyl phosphate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, and 2'-O-allyl-modified nucleotides. Nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, terminal nucleotides linked to cholesterol derivatives or dodecanoic acid di ...

[0148] For example, "2'-fluorinated nucleotides" refer to nucleotides in which the 2'-hydroxyl group of the ribosome is replaced by fluorine. "2'-O-methyl nucleotides" refer to nucleotides in which the 2'-hydroxyl group of the ribosome is replaced by a methoxy group. "2'-deoxy-modified nucleotides" refer to deoxyribonucleotides.

[0149] "A nucleotide containing a thiophosphate group" refers to a nucleotide in which one or more oxygen atoms on the phosphate group are replaced by sulfur atoms. "Modification of internucleotide thiophosphate bonding" refers to a modification in which two adjacent nucleotides are linked by a thiophosphate group.

[0150] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 5 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 5 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 5 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 5 (counting from the 3' end).

[0151] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 4 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 4 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 4 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 4 (counting from the 3' end).

[0152] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 3 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 3 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 3 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 3 (counting from the 3' end).

[0153] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 and 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 and 2 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkages modifications at positions 1 to 2 (counting from the 3' end).

[0154] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 2 (counting from the 3' end), wherein the 3' end of the sense strand is linked to a ligand moiety, the 3' end nucleotide of the sense strand is linked to the ligand moiety by a phosphorothioate linkage.

[0155] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 2 (counting from the 3' end), wherein the 3' end of the sense strand is linked to a ligand moiety, the 3' end nucleotide of the sense strand is linked to the ligand moiety by a phosphorothioate linkage.

[0156] As used herein, a "ligand moiety" refers to a chemical moiety conjugated to a dsRNA (particularly an siRNA) that is capable of altering the distribution, targeting, or lifetime of the dsRNA (particularly an siRNA). In some embodiments, the ligand moiety 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 the ligand moiety. In some embodiments, the ligand moiety targets the asialoglycoprotein receptor (ASGPR) on hepatocytes. Binding of the ligand moiety to the ASGPR mediates internalization via clathrin-coated vesicles. Maturation of the endosome results in a decrease in the pH of the lysosome, which promotes dissociation of the ligand-receptor complex, thereby releasing the dsRNA (particularly an siRNA). Conjugation of the ligand moiety that targets the asialoglycoprotein receptor (ASGPR) on hepatocytes results in efficacy and stability of the dsRNA (particularly an siRNA) in vivo or in a cell. This facilitates subcutaneous administration of the dsRNA (particularly an siRNA).

[0157] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," and other similar terms, and includes inhibition at any level.

[0158] The phrase "inhibiting expression of an LPA gene" 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.

[0159] "Inhibiting expression of an LPA gene" includes inhibition of an LPA gene at any level, e.g., at least partial inhibition 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, e.g., mRNA level of apolipoprotein(a), protein level of apolipoprotein(a). The level can be assessed in a single cell or in a group of cells, including, e.g., a sample derived from a subject.

[0160] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with LPA gene expression compared to a control level. The control level can be any type of control level utilized in the art, e.g., a pre-dosing 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.

[0161] "Hydroxyl protecting group" refers to a group of atoms that can prevent a hydroxyl group from undergoing a chemical reaction, yet can be removed to restore the hydroxyl group under specified conditions. It mainly includes silyl-type protecting group, acyl-type protecting group or ether-type protecting group, preferably the following: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM).

[0162] "Halo" or "halogen" means fluoro (F), chloro (CI), bromo (Br), and iodo (I).

[0163] "C 1-6 "Haloalkyl" means a "C 1-6 alkyl" group as described above, substituted by one or more halogen groups. In some embodiments, C 1-4 haloalkyl groups are particularly preferred, more preferably C 1-2 haloalkyl groups. Exemplary haloalkyl groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCI3, -CH2CI, -CHCI2, 2,2,2-trifluoro-l,l-dimethyl-ethyl, and the like. Haloalkyl groups can be substituted at any available attachment point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0164] "C 1-6 "Alkylene" means a divalent radical, derived by the removal of one hydrogen from a "C 1-6 alkyl" group, and can be substituted or unsubstituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene, and C 1-2 alkylene groups are preferred. Unsubstituted alkylene groups include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylene groups, for example, alkylene groups substituted by one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like. "C 1-2 "Alkylene" means a divalent radical, derived by the removal of one hydrogen from a "C 1-2 alkyl" group, and can be substituted or unsubstituted. In some embodiments, "C 1-2"Alkylene" is methylene (-CH2-), or ethylene (-CH2CH2-), or substituted methylene or substituted ethylene. "Substituted methylene" can be -CH(CH3)-, or -C(CH3)2-, etc. "Substituted ethylene" can be -CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, or -CH2C(CH3) 2- etc.

[0165] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating or expressing another nucleic acid to which it is linked. The "vector" herein is generally a plasmid, a virus, or a phage which can replicate in a host cell autonomously, it is preferably a multi-copy vector. In addition, the vector usually has a marker which allows the selection of transformed cells, such as an antibiotic resistance gene, etc. In addition, the vector can have a promoter and / or terminator for the expression of the introduced gene. The vector can be, for example, a vector derived from a bacterial plasmid, a viral vector, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, a phagemid, etc.

[0166] As used herein, the terms "treat," "treatment," and the like, refer to administering an agent or performing a procedure in order to effect an outcome. These outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include the treatment of a disease or condition (e.g., cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual that is predisposed or does not yet experience the disease (e.g., including a disease that can be associated with or caused by a primary disease); (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing the partial or complete regression of the disease. Treatment can refer to any indicia of success in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as elimination; reduction in severity; reduction in recurrence rate; or an increase in the rate of remission. Treatment or amelioration of a symptom is based on one or more objective or subjective parameters; including the results of a physician's examination. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or ameliorate a symptom or condition associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0167] The term "effective amount" as used herein refers to an amount which, when administered to a subject for treating a disease, is sufficient to effect such treatment of the disease.

[0168] As used herein, the term "subject" refers to any mammalian subject in whom diagnosis, cure, mitigation, or treatment is desired. "Mammal" for purposes of treatment includes humans, domestic and farm animals, and laboratory and sports animals, e.g., dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

[0169] I. dsRNA

[0170] The present application provides a double-stranded RNA (dsRNA) for inhibiting LPA gene expression, the dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82.

[0171] In some embodiments, the double-stranded region formed by the sense strand and the antisense strand is fully complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary, wherein it can comprise 1, 2, 3, 4, or 5 non-complementary sites.

[0172] In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41.

[0173] In some embodiments, the sense strand and the antisense strand are each independently 15-27 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 nucleotides, preferably 18-25 nucleotides, more preferably 19-21 nucleotides.

[0174] In some embodiments, the double-stranded region is 15-25 nucleotide pairs in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotide pairs, preferably 16-23 nucleotide pairs, more preferably 18-20 nucleotide pairs.

[0175] In some embodiments, the dsRNA of the present application is an siRNA. In other embodiments, a hairpin loop is formed between the sense strand and the antisense strand of the dsRNA of the present application.

[0176] In some embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 1 nucleotide. In some particular embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 1 nucleotide. In some particular embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides. In some particular embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 3 nucleotides. In some particular embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 4 nucleotides.

[0177] In some particular embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 1 nucleotide. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 3 nucleotides. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 4 nucleotides. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least 2 nucleotides.

[0178] Preferably the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides.

[0179] In some embodiments, the sense strand and the antisense strand are of the same length.

[0180] In some embodiments, the full length of the sense strand is complementary to the full length of the antisense strand forming a double strand, i.e. having blunt ends.

[0181] In other embodiments, the sense strand and the antisense strand are of the same length, a portion of the sense strand is complementary to a portion of the antisense strand, i.e. both the sense strand and the antisense strand have a 5' overhang. In some embodiments, the sense strand and the antisense strand are of different lengths. In preferred embodiments, the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably 2 or 3 nucleotides.

[0182] The dsRNAs of the application include dsRNAs having a nucleotide overhang at one end (i.e., having one overhang and one blunt end) or having a nucleotide overhang at both ends. For example, the 5 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the sense strand comprises an overhang of one or more nucleotides. For example, the 5 '-end of the antisense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the antisense strand comprises an overhang of one or more nucleotides. For example, the 5 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 5 '-end of the antisense strand comprises an overhang of one or more nucleotides. For example, the 3 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the antisense strand comprises an overhang of one or more nucleotides. For example, the 5 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the sense strand comprises a blunt end. For example, the 3 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the sense strand comprises a blunt end. For example, the 5 '-end of the antisense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the antisense strand comprises a blunt end. For example, the 3 '-end of the antisense strand of the dsRNA comprises an overhang of one or more nucleotides and the 5 '-end of the antisense strand comprises a blunt end.

[0183] In some preferred embodiments, the 3 '-end of the antisense strand of the dsRNA of the application comprises an overhang of one or more nucleotides and the 5 '-end of the antisense strand comprises a blunt end. In some more preferred embodiments, the 3 '-end of the antisense strand of the dsRNA of the application comprises an overhang of 1, 2, 3, or 4 nucleotides and the 5 '-end of the antisense strand comprises a blunt end. In some more preferred embodiments, the 3 '-end of the antisense strand of the dsRNA of the application comprises an overhang of 2 nucleotides and the 5 '-end of the antisense strand comprises a blunt end.

[0184] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, or at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82, preferably the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82.

[0185] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, or a nucleotide sequence of at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41, preferably the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41.

[0186] In some embodiments, the dsRNA comprises any one of the paired sense strand sequence and antisense strand sequence as set forth in Table 3.

[0187] II. Modifications of Nucleotides

[0188] In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, at least 95% of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the antisense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, at least 95% of the nucleotides of the sense strand are modified nucleotides.

[0189] In some embodiments, all of the nucleotides of the sense strand are modified nucleotides and / or all of the nucleotides of the antisense strand are modified nucleotides.

[0190] The modifications of nucleotides described herein can be modifications on the phosphate group, the ribose group, and / or the base group of the nucleotide.

[0191] In some particular embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of 2'-0-alkyl modified nucleotides (e.g., 2'-0-methyl modified nucleotides), 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides comprising phosphorothioate groups, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-0-allyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases, terminal nucleotides linked to a cholesteryl derivative or dodecanoic acid didecanoyl amide group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, glycol modified nucleotides (GNAs), and 2-0-(N-methylacetamide) modified nucleotides.

[0192] In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, and nucleotides comprising phosphorothioate groups. In some preferred embodiments, the sense strand and / or the antisense strand comprises at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprises at least 8 2'-0-methyl modified nucleotides. In some preferred embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate internucleotide linkages, preferably 2-3 phosphorothioate internucleotide linkages. In some embodiments, the sense strand and / or antisense strand comprises adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides, and / or cytosine deoxyribonucleotides. In more preferred embodiments, the sense strand and / or antisense strand comprises thymine deoxyribonucleotides. In most preferred embodiments, the sense strand comprises thymine deoxyribonucleotides.

[0193] In some preferred embodiments, the antisense strand comprises any one of the nucleotide sequences set forth in Table 5, and / or the sense strand comprises any one of the modified nucleotide sequences set forth in Table 4A. In some preferred embodiments, the dsRNA comprises the paired sense strand sequence and antisense strand sequence set forth in any one of Table 6.

[0194] III. Ligand Moieties

[0195] The dsRNA described herein is further conjugated to a ligand moiety, which preferably comprises N-acetylgalactosamine. In preferred embodiments, the sense strand of the dsRNA is conjugated to the ligand moiety. In some embodiments, the 3' end of the sense strand is conjugated to the ligand moiety. In other embodiments, the 5' end of the sense strand is conjugated to the ligand moiety.

[0196] In some embodiments, the ligand moiety comprises a conjugation group according to Formula (X'):

[0197] wherein,

[0198] represents the position of attachment to the dsRNA;

[0199] Q is independently H,

[0200] wherein L1is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0201] L2is a bond or -CH2CH2C(O)-;

[0202] L3is a bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b , or -C(O)CH2-;

[0203] L4is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c , or -NHC(O)-(CH2) d -;

[0204] wherein a = 0, 1, 2, or 3;

[0205] b = 1, 2, 3, 4, or 5;

[0206] c = 1, 2, 3, 4, or 5;

[0207] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0208] L is a bond, -CH2O-, or -NHC(O)-;

[0209] L' is a bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O) e -;

[0210] wherein e is 1, 2, 3, 4, or 5;

[0211] T is a bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0212] wherein M is

[0213] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0214] or R1and R3together form -C 1-2 alkylene-, and R2is H;

[0215] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0216] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0217] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0218] In some embodiments, the conjugate group is represented by Formula (I'):

[0219] wherein,

[0220] represents the position of attachment to the dsRNA;

[0221] Q is independently H,

[0222] wherein L1is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0223] L2is a bond or -CH2CH2C(O)-;

[0224] L3is a bond, -(NHCH2CH2) b-、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0225] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or -NHC(O)-(CH2) d -;

[0226] Where a = 0, 1, 2 or 3;

[0227] b = 1, 2, 3, 4 or 5;

[0228] c = 1, 2, 3, 4 or 5;

[0229] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0230] L is either -CH2O- or -NHC(O)-;

[0231] L' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0232] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0233] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0234] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0235] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0236] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0237] In some specific implementation plans, where,

[0238] Q is independently of H or

[0239] Where L1 is either -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0240] L2 is -CH2CH2C(O)-;

[0241] L3 is -(NHCH2CH2) b - or -(NHCH2CH2CH2) b -;

[0242] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -;

[0243] wherein a = 0, 1, 2 or 3;

[0244] b = 1, 2, 3, 4 or 5;

[0245] c = 1, 2, 3, 4 or 5;

[0246] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0247] L is -CH2O-;

[0248] L’ is a chemical bond;

[0249] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O- and R3 is H;

[0250] or R1 and R3 together form -C 1-2 alkylene-, and R2 is H;

[0251] wherein R is -OR’, -CH2OR’ or -CH2CH2OR’, wherein R’ is H, a hydroxyl protecting group or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4’-dimethoxytrityl;

[0252] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0253] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0254] In some embodiments, the conjugate group is represented by formula (I’-1), formula (I’-2) or formula (I’-3):

[0255] wherein,

[0256] represents the position of attachment to the dsRNA;

[0257] Q is

[0258] L1is -CH2O- or -NHC(O)-;

[0259] L2is -CH2CH2C(O)-;

[0260] L3is -(NHCH2CH2) b - or -(NHCH2CH2CH2) b -;

[0261] L4is -(OCH2CH2) c - or -NHC(O)-(CH2) d -;

[0262] wherein b = 1, 2, 3, 4 or 5;

[0263] c = 1, 2, 3, 4 or 5;

[0264] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0265] L is -CH2O-;

[0266] R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'- dimethoxytrityl, or a solid support;

[0267] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0268] In some specific embodiments, wherein,

[0269] Q is independently H,

[0270] L1is -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0271] L2is -CH2CH2C(O)-;

[0272] L3is -(NHCH2CH2) b -, -(NHCH2CH2CH2) b - or -C(O)CH2-;

[0273] L4is -(OCH2CH2) c - or -NHC(O)-(CH2) d -;

[0274] wherein a = 0, 1, 2 or 3;

[0275] b = 1, 2, 3, 4 or 5;

[0276] c = 1, 2, 3, 4 or 5;

[0277] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0278] L is either -CH2O- or -NHC(O)-;

[0279] L' represents a chemical bond or -C(O)NH-;

[0280] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0281] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0282] Wherein R is -OR', -CH2OR' or -CH2CH2OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl;

[0283] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0284] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0285] In some embodiments, the conjugating group is as shown in formula (II'-1) or formula (II'-2):

[0286] in,

[0287] Indicates the location where it is linked to dsRNA;

[0288] Q independently for

[0289] Where L1 is -CH2O- or -CH2O-CH2CH2O-;

[0290] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b -or-C(O)CH2-;

[0291] L4 is -(OCH2CH2) c -or -NHC(O)-(CH2) d -;

[0292] wherein b = 1, 2, 3, 4, or 5;

[0293] c = 1, 2, 3, 4, or 5;

[0294] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0295] L is -NHC(O)-;

[0296] L' is a bond or -C(O)NH-;

[0297] R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'- dimethoxytrityl, or a solid support;

[0298] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0299] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0300] In some specific embodiments, wherein,

[0301] Q is independently H,

[0302] wherein L1is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0303] L2is a bond;

[0304] L3is -(NHCH2CH2) b -, -(NHCH2CH2CH2) b , or -C(O)CH2-;

[0305] L4is -(OCH2CH2) c , or -NHC(O)-(CH2) d -;

[0306] wherein a = 0, 1, 2, or 3;

[0307] b = 1, 2, 3, 4, or 5;

[0308] c = 1, 2, 3, 4, or 5;

[0309] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0310] L is -CH2O- or -NHC(O)-;

[0311] L' is a bond or -C(O)NH-;

[0312] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0313] or R1and R3together form -C 1-2 alkylene-, and R2is H;

[0314] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0315] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0316] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0317] In some embodiments, wherein the conjugate group is represented by formula (II'-2):

[0318] wherein,

[0319] represents the position of attachment to the dsRNA;

[0320] Q is independently

[0321] wherein L1is -CH2- or -C(O)-;

[0322] L3is -(NHCH2CH2) b -;

[0323] L4is -(OCH2CH2) c -;

[0324] wherein b = 1, 2, 3, 4, or 5;

[0325] c = 1, 2, 3, 4, or 5;

[0326] L is -CH2O- or -NHC(O)-;

[0327] R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0328] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0329] In some specific embodiments, wherein:

[0330] Q is independently H,

[0331] wherein L1is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0332] L2is a bond or -CH2CH2C(O)-;

[0333] L3is a bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b , or -C(O)CH2-;

[0334] L4is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -;

[0335] wherein a = 0, 1, 2, or 3;

[0336] b = 1, 2, 3, 4, or 5;

[0337] c = 1, 2, 3, 4, or 5;

[0338] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0339] L is a bond, -CH2O-, or -NHC(O)-;

[0340] L' is a bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O) e -;

[0341] wherein e is 1, 2, 3, 4, or 5;

[0342] T is a bond, -CH2-, -M-, -CH2-M-, or -C(O)-M-;

[0343] wherein M is

[0344] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0345] or R1and R3together form -C 1-2 alkylene-, and R2is H;

[0346] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0347] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0348] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0349] In some specific embodiments, wherein,

[0350] T is -M-, -CH2-M-, or -C(O)-M-, wherein M is

[0351] In some specific embodiments, wherein,

[0352] Q is independently H or

[0353] wherein L1is -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0354] L2is -CH2CH2C(O)-;

[0355] L3is -(NHCH2CH2) b or -(NHCH2CH2CH2) b -;

[0356] L4is -(OCH2CH2) c or -NHC(O)-(CH2) d -;

[0357] wherein a = 0, 1, 2, or 3;

[0358] b = 1, 2, 3, 4, or 5;

[0359] c = 1, 2, 3, 4, or 5;

[0360] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0361] L is a chemical bond or -CH2O-;

[0362] L' is a chemical bond or -O(CH2CH2O) e -;

[0363] wherein e is 1, 2, 3, 4 or 5;

[0364] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0365] or R1and R3together form -C 1-2 alkylene-, and R2is H;

[0366] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0367] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0368] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0369] wherein T is as defined in the above embodiments.

[0370] In some embodiments, wherein the conjugate group is as shown in formula (III'-1), formula (III'-2) or formula (III'-3):

[0371] wherein,

[0372] Q is

[0373] wherein L1is -CH2O- or -NHC(O)-;

[0374] L2is -CH2CH2C(O)-;

[0375] L3is -(NHCH2CH2) b or -(NHCH2CH2CH2) b -;

[0376] L4is -(OCH2CH2) c or -NHC(O)-(CH2) d -;

[0377] wherein b = 1, 2, 3, 4 or 5;

[0378] c = 1, 2, 3, 4 or 5;

[0379] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0380] L is a chemical bond or -CH2O-;

[0381] wherein R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, or a solid support;

[0382] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0383] wherein T is as defined in the above embodiments.

[0384] In some specific embodiments, wherein,

[0385] Q is independently H,

[0386] wherein L1is -CH2-, -CH2O-, or -C(O)-;

[0387] L2is a chemical bond;

[0388] L3is -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0389] L4is -(OCH2CH2) c - or -NHC(O)-(CH2) d -;

[0390] wherein b = 1, 2, 3, 4, or 5;

[0391] c = 1, 2, 3, 4, or 5;

[0392] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0393] L is a chemical bond or -NHC(O)-;

[0394] L' is a chemical bond;

[0395] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0396] or R1and R3together form -C 1-2 alkylene-, and R2is H;

[0397] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0398] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0399] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0400] wherein T is as defined in the above embodiments.

[0401] In some embodiments, wherein the conjugate group is as shown in Formula (IV-1) or Formula (IV-2):

[0402] wherein,

[0403] Q is independently

[0404] wherein L1is -CH2-, -CH2O-, or -C(O)-;

[0405] L3is -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0406] L4is -(OCH2CH2) c -, or -NHC(O)-(CH2) d -;

[0407] wherein b = 1, 2, 3, 4, or 5;

[0408] c = 1, 2, 3, 4, or 5;

[0409] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0410] L is a chemical bond or -NHC(O)-;

[0411] L' is a chemical bond;

[0412] wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0413] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0414] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0415] wherein T is as defined in the embodiments above.

[0416] In some specific embodiments, wherein:

[0417] Q is independently H,

[0418] wherein L1is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0419] L2is a bond or -CH2CH2C(O)-;

[0420] L3is a bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b , or -C(O)CH2-;

[0421] L4is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -;

[0422] wherein a = 0, 1, 2, or 3;

[0423] b = 1, 2, 3, 4, or 5;

[0424] c = 1, 2, 3, 4, or 5;

[0425] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0426] L is a bond, -CH2O-, or -NHC(O)-;

[0427] L' is -O(CH2CH2O) e -;

[0428] wherein e is 1, 2, 3, 4, or 5;

[0429] T is a bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0430] wherein M is

[0431] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0432] or R1and R3together form -C 1-2 alkylene-, and R2is H;

[0433] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0434] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0435] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0436] In some preferred embodiments, wherein the conjugate group is selected from Table 1 and Table 2. In some embodiments, the ligand targets an asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand targets an asialoglycoprotein receptor (ASGPR) on a hepatocyte.

[0437] In some embodiments, wherein the ligand has the following structure:

[0438] wherein represents the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, more preferably the 3' end of the sense strand of the dsRNA, via a phosphonate group or a thiophosphonate group.

[0439] In some embodiments, wherein the ligand has the following structure:

[0440] wherein represents the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, more preferably the 3' end of the sense strand of the dsRNA, via a phosphonate group or a thiophosphonate group.

[0441] In some embodiments, wherein the ligand has the following structure:

[0442] wherein represents the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, more preferably the 3' end of the sense strand of the dsRNA, via a phosphonate group or a thiophosphonate group.

[0443] In some embodiments, wherein the ligand has the following structure:

[0444] wherein denotes the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, more preferably the 3' end of the sense strand of the dsRNA, via a phosphonate group or a thiophosphonate group.

[0445] In some embodiments, the sense strand of the dsRNA of the disclosure comprises any one of the nucleotide sequences set forth in Specification Table 4B.

[0446] In some embodiments, the dsRNA of the disclosure comprises any one pair of paired sense strand sequence and antisense strand sequence set forth in Specification Table 6B.

[0447] In some specific embodiments, the dsRNA of the disclosure has the following structure:

[0448] (1) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 121), and the antisense strand comprises (SCP-U) sAfsUmAmAmCmUfCmUmGmUmCfCmAfUmUfAmCmCmsGmsUm (SEQ ID NO: 342);

[0449] (2) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 122), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsGmsUm (SEQ ID NO: 305);

[0450] (3) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAm (SEQ ID NO: 123), and the antisense strand comprises (SCP-U) sdAsUmCmdAAmdGCmCmAmGmdCAmUfUmUmGmGmGmsUmsAm (SEQ ID NO: 317);

[0451] (4) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAm (SEQ ID NO: 124), and the antisense strand comprises (SCP-U) sAfsUmCmAmAfGmCmCmAmGmCfAmUfUmUfGmGmGmsUmsAm (SEQ ID NO: 309);

[0452] (5) the sense strand comprises UmsGmsGmUmAmAmUfGfGfAmCmAmGmAmGmUmUmAmsAm (SEQ ID NO: 125), and the antisense strand comprises (SCP-U) sUfsAmAmCmUmCfUmGmUmCmCfAmUfUmAfCmCmAmsUmsGm (SEQ ID NO: 340);

[0453] (6) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAm (SEQ ID NO: 126), and the antisense strand comprises (SCP-U) sdAsGmAmdTGmdACmCmAmAmdGCmUfUmGmGmCmAmsAmsGm (SEQ ID NO: 334);

[0454] (7) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAm (SEQ ID NO: 127), and the antisense strand comprises (SCP-U) sAfsGmAmUmGmAfCmCmAmAmGfCmUfUmGfGmCmAmsAmsGm (SEQ ID NO: 314);

[0455] (8) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 128), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsCmsUm (SEQ ID NO: 345);

[0456] (9) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 129), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsAmsUm (SEQ ID NO: 346); or

[0457] (10) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 130), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsUmsUm (SEQ ID NO: 347).

[0458] In a preferred embodiment, the 3' end of the sense strand of the dsRNA is conjugated to a ligand moiety having the structure

[0459] wherein represents the position of attachment to the 3' end of the sense strand of the dsRNA via a phosphorothioate group.

[0460] IV. Inhibition of LPA gene expression

[0461] The dsRNAs of the application are capable of inhibiting LPA gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0462] Inhibition of LPA gene expression can be manifested by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present in, for example, a sample derived from a subject) in which the LPA gene is transcribed and which cell or cells has been treated (for example, by contacting the cell or cells with a dsRNA of the application, or by administering a dsRNA of the application to a subject in which the cells now or formerly exist), such that LPA gene expression is inhibited as compared to a second cell or group of cells (control cell(s)) that is / are substantially identical to the first cell or group of cells but has / have not been so treated.

[0463] In preferred embodiments, the inhibition is assessed by expressing the level of mRNA in the treated cell as a percentage of the level of mRNA in a control cell using the following formula: -△△CtValues are expressed as differences between experimental and control groups, where ΔΔCt = [(Ct experimental gene of interest - Ct experimental internal control) - (Ct control gene of interest - Ct control internal control)].

[0464] Alternatively, inhibition of LPA gene expression, e.g., apolipoprotein(a) protein expression, can be assessed in terms of a decrease in a parameter that is functionally related to LPA gene expression. LPA gene silencing can be determined in any cell that expresses LPA constitutively or by genome engineering and by any assay known in the art.

[0465] Inhibition of apolipoprotein(a) protein expression can be manifested by a decrease in the level of apolipoprotein(a) protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above with respect to assessment of mRNA inhibition, inhibition of the level of protein expression in a treated cell or group of cells can similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

[0466] Control cells or groups of cells that can be used to assess inhibition of LPA gene expression include cells or groups of cells that have not been contacted with a dsRNA of the application. For example, the control cells or groups of cells can be derived from the individual subject (e.g., a human or animal subject) prior to treatment of the subject with a dsRNA.

[0467] V. Off-target effects

[0468] The LPA gene encoding apolipoprotein(a) is highly similar in sequence to the plasminogen-encoding gene, PLG, and is prone to off-target effects. The LPA gene encoding apolipoprotein(a) has evolved from the plasminogen gene over thousands of years of replication and reshaping for unknown etiological and physiological reasons. Plasminogen contains five kringle (KI to KV) and one protease domain. Apolipoprotein(a) does not contain KI to KIII of plasminogen, but contains 10 isoforms of KIV (one copy of KIV1 and KIV3-10, 1 copy to >40 copies of KIV2), one copy of KV, and one inactive protease domain. Apolipoprotein(a) is hydrophilic and can bind to denuded and exposed lysine-rich vascular endothelium, similar to plasminogen and in competition with it. Therefore, off-target effects, particularly off-target effects against PLG, need to be considered while designing siRNAs against the LPA gene.

[0469] In some embodiments of the present invention, the dsRNA (e.g., siRNA) used to inhibit LPA gene expression is able to specifically bind to LPA mRNA, but not bind to or bind to PLG mRNA with a lower affinity, thereby causing a more specific reduction in LPA gene expression, without reducing or only negligibly reducing PLG gene expression, thus achieving a lower off-target effect.

[0470] VI. Carrier

[0471] This invention provides a vector containing a nucleotide sequence encoding the dsRNA described herein. The vector of this invention is capable of amplifying and / or expressing the nucleotide encoding the dsRNA linked thereto. The vector of this invention can be a virus, plasmid, or granule.

[0472] The dsRNA of the LPA-targeting mRNA can be expressed from transcriptional units inserted into DNA or RNA vectors. Expression can be transient (lasting hours to weeks) or persistent (lasting weeks to months or longer), depending on the specific construct used and the type of target tissue or cell. The coding nucleotides of the LPA-targeting mRNA dsRNA can be introduced into linear constructs, circular plasmids, or viral vectors. The nucleotides encoding the LPA-targeting mRNA dsRNA can be stably expressed by integration into the cellular genome or by stable extrachromosomal inheritance. Generally, dsRNA expression vectors are typically DNA plasmids or viral vectors. Viral vector systems containing the coding sequence of the LPA-targeting mRNA dsRNA include, but are not limited to: (a) adenovirus vectors; (b) retrovirus vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) microRNA virus vectors; or (i) poxvirus vectors.

[0473] VII. Cells

[0474] This invention provides cells containing the dsRNA or vector described herein.

[0475] VIII. Pharmaceutical Compositions

[0476] The present invention provides pharmaceutical compositions comprising the dsRNA, vector or cell described herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0477] As used in this article, "pharmaceutically acceptable" means compounds, materials, compositions, and / or dosage forms that, to the extent of proper medical judgment, are suitable for contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic reactions, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio.

[0478] In the present context, a pharmaceutically acceptable carrier means a pharmaceutical carrier which facilitates administration of the dsRNA or a vector or cell comprising a coding sequence thereof to the human body and / or facilitates its absorption or effects. For example: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; humectants such as glycerol; disintegrants such as agar agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorptive carriers such as kaolin and bentonite clay; lubricants such as talc, calcium / magnesium stearate, polyethylene glycol, etc. Other adjuvants such as flavourings, sweeteners, etc. can also be added to the composition.

[0479] The pharmaceutical composition of the present application can comprise a pharmaceutically acceptable diluent or a sustained release matrix into which the dsRNA or vector of the present application is embedded.

[0480] The pharmaceutical composition of the present application can comprise a drug delivery system for delivery of the dsRNA. The drug delivery system of the present application includes, but is not limited to, nanoparticles (e.g. lipid nanoparticles, polymer-based nanoparticles), polymers, PEG, or cationic delivery systems, polylactic acid (PLA) microspheres, poly(lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, lipid microtubules, cholesterol, PEG lipid PEG-2000-C-DMG, PEG-2000-DMG (Moderna), ALC-0159 or DSPC.

[0481] In some embodiments, the siRNA or vector in the pharmaceutical composition of the present application can be comprised in polymers and polymer-based nanoparticles.

[0482] In some specific embodiments, the polymer is a poly(lactic-co-glycolic acid) or PLGA-based polymer. In some specific embodiments, the PLGA-based polymer is engineered to contain a cationic group alone.

[0483] In some specific embodiments, the polymer contains amine groups that can become cationic, such as polyethylenimine (PEI) and poly(L-lysine) (PLL), which are capable of forming complexes with siRNA through electrostatic interactions and deliver siRNA into cells. In some embodiments, PEG and PLL are chemically modified to improve in vivo efficacy and tolerability.

[0484] In some embodiments, the siRNA, vector, or cell in a pharmaceutical composition of the application can be delivered by a cationic polymer, poly(beta-amino ester) (PBAE).

[0485] IX. Kits

[0486] The present application provides a kit comprising a dsRNA, vector, or cell described herein.

[0487] The present application also provides a kit for using a dsRNA and / or performing a method of the present application. Such a kit includes one or more dsRNAs, vectors, or cells described herein, and can further include instructions for use. The instructions for use can include instructions for inhibiting LPA gene expression in a cell by contacting the cell with a dsRNA or vector described herein in an amount effective to inhibit LPA gene expression.

[0488] In the case where a dsRNA or vector described herein is contacted with a cell in vitro, optionally, a kit of the present application can further include a means for contacting a cell with a dsRNA or vector described herein (e.g., an injection device) or a means for measuring the inhibitory effect on the LPA gene (e.g., a device for measuring inhibition of LPA mRNA or protein). Such a device for measuring inhibition of the LPA gene can include a device for obtaining a sample (e.g., a plasma sample) from a subject.

[0489] In the case where a dsRNA, vector, or cell that has been introduced with a dsRNA or vector in vitro is administered in vivo, a kit of the present application can further optionally include a device for administering a dsRNA, vector, or cell described herein to a subject or a device for determining a therapeutically effective amount or a prophylactically effective amount.

[0490] X. Therapeutic methods, pharmaceutical uses

[0491] The present application provides a method of reducing apolipoprotein(a) in a subject, the method comprising the step of administering to the subject a dsRNA, vector, cell, or pharmaceutical composition described herein.

[0492] The present application provides a method of treating, preventing, inhibiting, or ameliorating a disease or disorder in a subject that benefits from a reduction in LPA gene expression, the method comprising the step of administering to the subject a dsRNA, vector, cell, or pharmaceutical composition described herein. The present application also provides a method of treating, preventing, inhibiting, or ameliorating at least one symptom in a patient having a disease or disorder that benefits from a reduction in LPA gene expression.

[0493] In some embodiments, the disease or disorder that benefits from a reduction in LPA gene expression is an apolipoprotein(a)-related disease.

[0494] In some embodiments, the method of reducing apolipoprotein(a) in a subject, the method of treating, preventing, inhibiting or ameliorating a disease or disorder that benefits from a reduction in LPA gene expression in a subject, or the method of treating, preventing, inhibiting or ameliorating at least one symptom in a patient having a disease or disorder that benefits from a reduction in LPA gene expression, of the present application comprises subcutaneously administering, topically administering, or intravenously administering the dsRNA, vector, cell or pharmaceutical composition to the subject. In some embodiments, the subject is a human patient.

[0495] The present application also relates to a dsRNA, vector, cell or pharmaceutical composition described herein for use in reducing apolipoprotein(a) in a subject. The present application also relates to a dsRNA, vector, cell or pharmaceutical composition described herein for use in treating a disease associated with apolipoprotein(a) in a subject. The present application also relates to a dsRNA, vector, cell or pharmaceutical composition described herein for use in treating, preventing, inhibiting or ameliorating a disease or disorder that benefits from a reduction in LPA gene expression in a subject. The present application also relates to a dsRNA, vector, cell or pharmaceutical composition described herein for use in treating, preventing, inhibiting or ameliorating at least one symptom in a patient having a disease or disorder that benefits from a reduction in LPA gene expression.

[0496] The present application also relates to the use of a dsRNA, vector, cell or pharmaceutical composition described herein for the manufacture of a medicament for treating, preventing, inhibiting or ameliorating a disease or disorder that benefits from a reduction in LPA gene expression in a subject. The present application also relates to the use of a dsRNA, vector, cell or pharmaceutical composition described herein for the manufacture of a medicament for treating a disease associated with apolipoprotein(a) in a subject. The present application also relates to the use of a dsRNA, vector, cell or pharmaceutical composition described herein for the manufacture of a medicament for reducing apolipoprotein(a) in a subject. The present application also relates to the use of a dsRNA, vector, cell or pharmaceutical composition described herein for the manufacture of a medicament for treating, preventing, inhibiting or ameliorating at least one symptom in a patient having a disease or disorder that benefits from a reduction in LPA gene expression.

[0497] Sequences

[0498] The RNA sequences provided by the present application target the human LPA gene (or target gene, target mRNA sequence, target sequence). The target LPA mRNA sequence is the gene as set forth in Genbank Accession No. NM_005577.4.

[0499] The RNA sequences and compounds provided by the present application are set forth in Tables 3-6 below.

[0500] Table 3. Nucleotide sequences of sense and antisense strands targeting LPA mRNA

[0501] Tables 4 and 5 show modified RNA sequences used in the present application, respectively.

[0502] Herein, the meaning of each abbreviation is as follows:

[0503] A, U, G, and C stand for natural adenosine ribonucleotides, uracil ribonucleotides, guanosine ribonucleotides, and cytosine ribonucleotides, respectively.

[0504] d stands for a deoxyribonucleotide adjacent to its right. For example, dA, dT, dG, and dC stand for adenosine deoxyribonucleotides, thymine deoxyribonucleotides, guanosine deoxyribonucleotides, and cytosine deoxyribonucleotides, respectively.

[0505] i stands for an inosine ribonucleotide.

[0506] m stands for a 2’-OCH3-modified nucleotide adjacent to its left. For example, Am, Um, Gm, and Cm stand for 2’-OCH3-modified A, U, G, and C, respectively.

[0507] f stands for a 2’-fluoro-modified nucleotide adjacent to its left. For example, Af, Uf, Gf, and Cf stand for 2’-fluoro-modified A, U, G, and C, respectively.

[0508] “s” or s- stands for two nucleotides adjacent to its left and right and / or the delivery vehicle through a phosphorothioate linkage.

[0509] VP stands for a vinylphosphonate-modified nucleotide adjacent to its right, which is well known in the art, see, e.g., PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419

[0510] GL6 stands for a GalNAc delivery vehicle of the following structure, wherein stands for the position of linkage to the dsRNA through a phosphonate or phosphorothioate group.

[0511] SCP stands for a nucleotide surrogate of the following structure, wherein Base can be any base, for example, SCP-U stands for Base being uracil.

[0512] (NAG25)s stands for a GalNAc delivery vehicle of the following structure, wherein indicates the position of attachment to the dsRNA.

[0513] A(Gal) indicates a GalNAc delivery carrier of the following structure, wherein indicates the position of attachment to the dsRNA.

[0514] invdA indicates an inverted deoxyadenosine nucleotide, i.e., a deoxyadenosine nucleotide (dA) is attached to the nucleotide to the left via a 3’-3’ linkage, see, e.g., PCT Publication No. WO2017059223A2, structure below.

[0515] MepU indicates a nucleotide surrogate of the following structure.

[0516] Table 4A. Modified siRNA sense strand sequences targeting LPA mRNA (ligand not attached)

[0517] Table 4B. Modified siRNA sense strand sequences targeting LPA mRNA (ligand attached)

[0518] Table 5. Modified siRNA antisense strand sequences targeting LPA mRNA

[0519] Table 6A. Paired siRNA sense and antisense strands targeting LPA mRNA (ligand not attached)

[0520] Table 6B. Paired siRNA sense and antisense strands targeting LPA mRNA (ligand attached)

[0521] Table 7 Sense and antisense strands of positive control siRNA

[0522] Table 8 Sense and antisense strands of positive control siRNA paired

[0523] The present application will be further illustrated by the following examples. It should be understood that the following examples are illustrative only and should not be taken in a limiting sense on the scope of the present application. Examples

[0524] The materials used in the examples are as follows, unless otherwise specified:

[0525] Huh7 cell line was purchased from Nanjing Kebai, item number CBP60202

[0526] Hep3B cell line was purchased from Nanjing Kebai, item number CBP60197

[0527] PHH cells were purchased from Shanghai Xuanyi, item number QYLF-HPMC

[0528] HEK293A cell line was purchased from Nanjing Kebai, item number CBP60436

[0529] Balb / c mice were from Zhejiang Weitong Lihua, item number Balb / c

[0530] Example 1 Preparation of compound E7

[0531] Compound E7 is a monomer with GL6 oligonucleotide, the structure of which is shown above. Its synthesis method can be found in, for example, PCT Publication No. WO2023143571A1.

[0532] Example 2 Preparation of siRNA

[0533] The siRNA of the present application is prepared using the solid-phase phosphoramidite method well known in the art. The specific method can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and is briefly described as follows.

[0534] 1.1 Synthesis of the sense strand (SS strand)

[0535] By the solid-phase phosphoramidite synthesis method, using a blank CPG solid-phase carrier as the starting cycle, the nucleoside monomers and / or delivery carrier monomers are connected one by one in the order of the nucleotide arrangement of the sense strand from 3'-5' direction. Each connection of a monomer includes four steps of deprotection, coupling, capping, oxidation or thio. The synthesis scale is 5 pmol of oligonucleic acid synthesis conditions as follows:

[0536] Commercially available 2'-F, 2'-O-methyl and other modified phosphoramidites were used. Nucleoside monomers were provided as 0.05 mol / L solutions in acetonitrile, and the same conditions were used for each step, i.e. temperature of 25 °C, deprotection with 3% trichloroacetic acid in dichloromethane, deprotection for 3 times; coupling reaction with 0.25 mol / L ETT in acetonitrile, coupling for 2 times; capping with 10% acetic anhydride in acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v), capping for 2 times; oxidation with 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v), oxidation for 2 times; thioation with 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v), thioation for 2 times.

[0537] 1.2 Synthesis of antisense strand (AS strand)

[0538] By solid-phase phosphoramidite synthesis method, blank CPG solid-phase carrier was used as the starting cycle, and nucleoside monomers were connected one by one in the order of antisense strand nucleotide arrangement from 3'-5' direction. Each connection of a nucleoside monomer included four steps of deprotection, coupling, capping, oxidation or thioation, and the synthesis conditions of 5 umol of oligonucleic acid of the antisense strand were the same as those of the sense strand.

[0539] 1.3 Purification and annealing of oligonucleic acid

[0540] 1.3.1 Aminolysis

[0541] The synthesized solid-phase carrier (sense strand or antisense strand) was added to a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) was added, and the reaction was carried out at 35 °C constant temperature water bath for 16 hours (or at 55 °C constant temperature water bath for 8 hours), then filtered, and the solid-phase carrier was washed with ethanol / water three times, 1 mL each time, and the filtrate was concentrated by centrifugation, and the crude product was purified.

[0542] 1.3.2 Purification

[0543] The method of purification and desalination is well known to those skilled in the art. For example, a strong anion filler column can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and pipetted. A gel filler purification column can be used for desalination, and the elution system is pure water.

[0544] 1.3.3 Annealing

[0545] The sense strand (SS strand) and the antisense strand (AS strand) were mixed according to Table 6 and Table 8 in a molar ratio of SS strand / AS strand = 1 / 1.05, heated to 70-95 °C in a water bath, kept for 3-5 minutes, and naturally cooled to room temperature, and the system was freeze-dried to obtain the siRNA compound product, wherein DR001480 and DR009426 in Table 8 are used as positive controls.

[0546] Example 3. Screening experiment for the activity of primary mouse liver cells (PMH)

[0547] transfection

[0548] Freshly isolated primary liver cells from mice transfected with the human LPA gene (purchased from Cyagen Biosciences Co., Ltd.), counted, plated in 96-well plates, 100 μL / well, 1 x 102 4 Cells / well

[0549] Transfection: Add 2 μL of the compound to 198 μL of Opti-MEM and mix well. Add 0.9 μL of RNAiMAX to 14.1 μL of Opti-MEM and mix well to obtain the diluted compound (the compound concentration is selected below). After incubation for 5 minutes, mix with 15 μL of the diluted compound, let stand at room temperature for 10 minutes, add to the corresponding well, and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0550] Real-time PCR

[0551] Following the protocol of the high-throughput cell RNA extraction kit (Shanghai Fushen, FSF0035-TS-96T), cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) and reverse transcribed (PrimeScript). TM After using the 1st Strand cDNA Synthesis Kit (Takara, 6210B), quantitative real-time PCR was performed.

[0552] ReferenceTaqMan TM A 20 μL system of Fast Advanced Master Mix (ABI, 4444965) was used for real-time PCR (ABI, QuantStudio3). The reaction program was as follows: (50℃, 2 min) x 1 cycle; (95℃, 20 s) x 1 cycle; (95℃, 1 s; 60℃, 24 s) x 40 cycles.

[0553] Table 9A. Primer Information

[0554] Data statistics 372

[0555] Calculate 2 -△△Ct The value is then converted to a percentage to obtain the remaining percentage;

[0556] △△Ct=[(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0557] The target gene is hLPA, and the internal reference is mGAPDH.

[0558] The siRNA compound cell line activity screening was carried out at a compound concentration of 0.1 nM, and the experimental results are shown in Table 9B.

[0559] Table 9B. PMH activity screening experimental results

[0560] Example 4. PMH cell line IC50 activity screening experiment

[0561] According to the method of Example 2, more siRNAs of the application were tested.

[0562] Table 10. PMH cell line IC50 activity screening experimental results

[0563] Example 5. Compound efficacy long-term validation of LPA transgenic mouse model

[0564] The LPA transgenic mice (purchased from Sanyei Biotechnology, male, 18-21 g, 6-8 weeks) were grouped according to the serum Apo(a) expression, and the drug dose was calculated according to the weight of each animal. Subcutaneous injection was used for single dose administration. Before the experiment, the siRNA conjugate was dissolved in 0.9% sodium chloride aqueous solution and diluted to the required solution concentration and volume (dose 2 mpk). The physiological saline and siRNA conjugate were administered at a volume of 5 mL / kg.

[0565] The mice were bled from the orbital venous plexus before administration (recorded as day 0), and at 7, 14 and 28 days after administration (the sampling time points were adjusted according to different experimental requirements). At each time point, the serum Apo(a) protein was detected by ELISA kit (Abeam, ab212165).

[0566] The experimental results are shown in Table 11.

[0567] Table 11. Compound efficacy long-term validation experiment results of LPA transgenic mouse model

[0568] Example 6. PMH cell line IC50 activity screening experiment

[0569] According to the method of Example 2, more siRNAs of the application were tested.

[0570] The inhibition rate was calculated using the following formula: Inhibition rate = 100% - remaining percentage. The experimental results are shown in the following table.

[0571] Table 12. Results of IC50 activity screening experiment of PMH cell line

[0572] Example 7. Compound efficacy and long-acting property verification of LPA transgenic mouse model

[0573] According to the method of Example 5, more siRNAs of the application were tested, and the experimental results are shown in the following table.

[0574] Table 13. Results of compound efficacy and long-acting property verification experiment of LPA transgenic mouse model

[0575] Example 8. Compound efficacy and long-acting property verification of LPA transgenic mouse model

[0576] According to the method of Example 5, more siRNAs of the application were tested, and the experimental results are shown in the following table.

[0577] Table 14. Results of compound efficacy and long-acting property verification experiment of LPA transgenic mouse model

[0578] Example 9. Cynomolgus monkey efficacy verification

[0579] Cynomolgus monkey single subcutaneous injection screening test was carried out in Suzhou Guochen Biological. Elution monkeys were used, 3 in each group. Animals received single subcutaneous administration, and the administration amount was 2 mg / kg, 0.5 mL / kg.

[0580] Blood was taken and serum was separated before administration (D0), 7 days after administration (D7, same below), D14, D21, D28, D35, D42, D56, D70 and D84.

[0581] The change of Lp(a) level was tested by immunoturbidimetry using a full-automatic biochemical analyzer. It was expressed in the form of knockdown percentage. Knockdown percentage = remaining percentage - 100%. The results are shown in the following table.

[0582] Table 15. Cynomolgus monkey efficacy results (experiment 1)

[0583] Table 16. Cynomolgus monkey efficacy results (experiment 2)

[0584] The results show that the compounds of the application have comparable or better effects compared with the positive control DR001480.

Claims

1. A double-stranded nucleotide (dsRNA) for inhibiting LPA gene expression, the dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82, preferably the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 48, 50, 55, 68, 80, 81, and 82.

2. The dsRNA of claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41, preferably the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 14, 27, 39, 40, and 41.

3. The dsRNA of claim 1 or 2, wherein the dsRNA is an siRNA.

4. The dsRNA of any one of claims 1-3, wherein the double-stranded region is 15-25 nucleotide pairs in length, preferably 16-23 nucleotide pairs in length, more preferably 18-20 nucleotide pairs in length, most preferably 19 nucleotide pairs in length.

5. The dsRNA of any one of claims 1-4, wherein the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, or at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82, preferably the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, or at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 42-82, preferably the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, or at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 44, 48, 50, 55, 68, 80, 81, and 82.

6. The dsRNA of any one of claims 1-5, wherein the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41, preferably the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-41, preferably the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 14, 27, 39, 40, and 41. ​ Preferably, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, or a nucleotide sequence of at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 14, 27, 39, 40, and 41, or comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 7, 9, 14, 27, 39, 40, and 41.

7. The dsRNA of any one of claims 1-6, wherein the siRNA comprises any one of the paired sense strand sequence and antisense strand sequence as set forth in Table 3 of the specification, preferably, the siRNA comprises any one of the paired sense strand sequence and antisense strand sequence of: (1) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 3, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 44; (2) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 7, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 48; (3) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 9, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 50; (4) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 14, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 55; (5) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 27, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 68; (6) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 39, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 80; (7) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 40, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 81; or (8) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 41, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:

82.

8. The dsRNA of any one of claims 1-7, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. ​ ​ ​ ​ ​ ​ ​ ​ 9. The dsRNA of claim 8, wherein each of the sense strand and the antisense strand independently comprises one or more modified nucleotides selected from the group consisting of 2'-0-alkyl modified nucleotides (e.g., 2'-0-methyl modified nucleotides), SCP modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides comprising phosphorothioate groups, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-0-allyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases, terminal nucleotides linked to a cholesteryl derivative or dodecanoic acid didecanoyl amide group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, glycol modified nucleotides (GNAs), and 2-0-(N-methylacetamide) modified nucleotides.

10. The dsRNA of claim 9, wherein each of the sense strand and the antisense strand independently comprises one or more modified nucleotides selected from the group consisting of 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, SCP modified nucleotides, 2'-deoxy-modified nucleotides, and nucleotides comprising phosphorothioate groups.

11. The dsRNA of any one of claims 1-10, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) 2'-0-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and / or (ii) (counting from the 5' end) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21.

12. The dsRNA of any one of claims 1-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) 2'-0-methyl modified nucleotides at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21; (ii) (counting from the 5' end) a 2'-fluoro modified nucleotide at position 14; (iii) (counting from the 5' end) 2'-deoxy modifications at positions 2, 5, 7, and 12; (iv) (counting from the 5' end) an SCP modification at position 1; and / or (v) (counting from the 5' end) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21. ​ ​ ​ ​ ​ ​ (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21, counting from the 5' end.

13. The dsRNA of any one of claims 1-12, wherein the antisense strand has a length of 21 nucleotides and has (i) 2'-0-methyl modified nucleotides at positions 3, 5, 9, 11, 13, 15, 17, 19, 20, and 21, counting from the 5' end; (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18, counting from the 5' end; (iii) a GNA modification at position 7, counting from the 5' end; (iv) an SCP modification at position 1, counting from the 5' end; and / or (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21, counting from the 5' end.

14. The dsRNA of any one of claims 1-12, wherein the antisense strand has a length of 21 nucleotides and has (i) 2'-0-methyl modified nucleotides at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21, counting from the 5' end; (ii) 2'-fluoro modified nucleotides at positions 2, 7, 12, 14, and 16, counting from the 5' end; (iii) an SCP modified nucleotide at position 1, counting from the 5' end; and / or (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21, counting from the 5' end.

15. The dsRNA of any one of claims 1-12, wherein the antisense strand has a length of 21 nucleotides and has (i) 2'-0-methyl modified nucleotides at positions 1, 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21, counting from the 5' end; (ii) 2'-fluoro modified nucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end; (iii) an SCP modified nucleotide at position 1, counting from the 5' end; and / or (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21, counting from the 5' end.

16. The dsRNA of any one of claims 1-12, wherein the antisense strand has a length of 21 nucleotides and has (i) 2'-0-methyl modified nucleotides at positions 1, 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21, counting from the 5' end; (ii) 2'-fluoro modified nucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end; (iii) an SCP modified nucleotide at position 1, counting from the 5' end; and / or (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21, counting from the 5' end. (i) 2'-0-methyl modified nucleotides at positions (counting from the 5' end) 1, 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21; (ii) a 2'-fluoro modified nucleotide at position 14 (counting from the 5' end); (ii) 2'-deoxy modified nucleotides at positions 2, 5, 7, and 12 (counting from the 5' end); (iv) an SCP modified nucleotide at position 1 (counting from the 5' end); and / or (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end).

17. The dsRNA of any one of claims 1-16, wherein the sense strand has a length of 19 nucleotides and has: (i) 2'-0-methyl modified nucleotides at positions (counting from the 5' end) 1 to 6, 10 to 19, and 2'-fluoro modified nucleotides at positions 7-9; and / or (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 (counting from the 5' end).

18. The dsRNA of any one of claims 1-17, wherein the antisense strand comprises any one of the nucleotide sequences set forth in Table 5 of the specification, and / or the sense strand comprises any one of the nucleotide sequences set forth in Table 4A of the specification.

19. The dsRNA of claim 18, wherein the dsRNA comprises any one of the paired sense strand sequence and antisense strand sequence set forth in Table 6A of the specification.

20. The dsRNA of claim 19, wherein (1) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 121), and the antisense strand comprises (SCP-U)sAfsUmAmAmCmUfCmUmGmUmCfCmAfUmUfAmCmCmsGmsUm (SEQ ID NO: 342); (2) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 122), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsGmsUm (SEQ ID NO: 305); (3) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAm (SEQ ID NO: 123), and the antisense strand comprises (SCP-U)sdAsUmCmdAAmdGCmCmAmGmdCAmUfUmUmGmGmGmsUmsAm (SEQ ID NO: 317); (4) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAm (SEQ ID NO: 124), and the antisense strand comprises (SCP-U)sAfsUmCmAmAfGmCmCmAmGmCfAmUfUmUfGmGmGmsUmsAm (SEQ ID NO: 309); (5) the sense strand comprises UmsGmsGmUmAmAmUfGfGfAmCmAmGmAmGmUmUmAmsAm (SEQ ID NO: 125), and the antisense strand comprises (SCP-U)sUfsAmAmCmUmCfUmGmUmCmCfAmUfUmAfCmCmAmsUmsGm (SEQ ID NO: 340); (6) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAm (SEQ ID NO: 126), and the antisense strand comprises (SCP-U)sdAsGmAmdTGmdACmCmAmAmdGCmUfUmGmGmCmAmsAmsGm (SEQ ID NO: 334); (7) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAm (SEQ ID NO: 127), and the antisense strand comprises (SCP-U)sAfsGmAmUmGmAfCmCmAmAmGfCmUfUmGfGmCmAmsAmsGm (SEQ ID NO: 314); (8) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 128), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsCmsUm (SEQ ID NO: 345); (9) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 129), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsAmsUm (SEQ ID NO: 346); or (10) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAm (SEQ ID NO: 130), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsUmsUm (SEQ ID NO: 347).

21. The dsRNA of any one of claims 1-20, wherein the dsRNA is further conjugated to a ligand moiety, preferably the ligand moiety comprises N-acetylgalactosamine, preferably the 3' end of the sense strand is conjugated to the ligand moiety.

22. The dsRNA of claim 21, wherein the ligand has the structure: ###0002### wherein represents the position of attachment to the dsRNA, preferably the sense strand of the dsRNA, via a phosphoester group or a phosphorothioate group.

23. The dsRNA of claim 22, wherein the sense strand comprises any one of the nucleotide sequences shown in Table 4B of the specification.

24. The dsRNA of claim 23, wherein the dsRNA comprises any one of the paired sense strand sequence and antisense strand sequence shown in Table 6B of the specification, preferably the dsRNA comprises any one of the following paired sense strand sequence and antisense strand sequence: (1) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U) sAfsUmAmAmCmUfCmUmGmUmCfCmAfUmUfAmCmCmsGmsUm (SEQ ID NO: 342); (2) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsGmsUm (SEQ ID NO: 305); (3) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAms- GL6 (SEQ ID NO: 182), and the antisense strand comprises (SCP-U) sdAsUmCmdAAmdGCmCmAmGmdCAmUfUmUmGmGmGmsUmsAm (SEQ ID NO: 317); (4) the sense strand comprises CmsCmsCmAmAmAmUfGfCfUmGmGmCmUmUmGmAmUmsAms- GL6 (SEQ ID NO: 182), and the antisense strand comprises (SCP-U) sAfsUmCmAmAfGmCmCmAmGmCfAmUfUmUfGmGmGmsUmsAm (SEQ ID NO: 309); (5) the sense strand comprises UmsGmsGmUmAmAmUfGfGfAmCmAmGmAmGmUmUmAmsAms- GL6 (SEQ ID NO: 184), and the antisense strand comprises (SCP-U) sUfsAmAmCmUmCfUmGmUmCmCfAmUfUmAfCmCmAmsUmsGm (SEQ ID NO: 340); (6) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAms- GL6 (SEQ ID NO: 200), and the antisense strand comprises (SCP-U) sdAsGmAmdTGmdACmCmAmAmdGCmUfUmGmGmCmAmsAmsGm (SEQ ID NO: 334); (7) the sense strand comprises UmsGmsCmCmAmAmGfCfUfUmGmGmUmCmAmUmCmUmsAms- GL6 (SEQ ID NO: 200), and the antisense strand comprises (SCP-U) sAfsGmAmUmGmAfCmCmAmAmGfCmUfUmGfGmCmAmsAmsGm (SEQ ID NO: 314); (8) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsCmsUm (SEQ ID NO: 345); (9) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U) sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsAmsUm (SEQ ID NO: 346); or (10) the sense strand comprises GmsGmsUmAmAmUmGfGfAfCmAmGmAmGmUmUmAmUmsAms- GL6 (SEQ ID NO: 194), and the antisense strand comprises (SCP-U)sAfsUmAmAfCmUfCmUmGmUmCfCmAfUmUmAmCmCmsUmsUm (SEQ ID NO: 347).

25. A cell comprising the dsRNA of any one of claims 1-24.

26. A pharmaceutical composition comprising the dsRNA of any one of claims 1-24, or the cell of claim 25, and optionally a pharmaceutically acceptable carrier or excipient.

27. A kit comprising the dsRNA of any one of claims 1-24, the cell of claim 25, or the pharmaceutical composition of claim 26.

28. A method of treating a disease or disorder that benefits from a reduction in LPA gene expression in a subject, the method comprising the step of administering to the subject the dsRNA of any one of claims 1-24, the cell of claim 25, or the pharmaceutical composition of claim 26.

29. A method of preventing at least one symptom in a subject having a disease or disorder that benefits from a reduction in LPA gene expression, the method comprising the step of administering to the subject the dsRNA of any one of claims 1-24, the cell of claim 25, or the pharmaceutical composition of claim 26.

30. The method of claim 28 or 29, wherein the disease or disorder that benefits from a reduction in LPA gene expression is an apolipoprotein(a)-mediated or apolipoprotein(a)-related disease.

31. The method of claim 30, wherein the apolipoprotein(a)-mediated or apolipoprotein(a)-related disease is selected from the group consisting of Buerger'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, hyperlipoproteinemia, atherosclerosis (e.g., cerebrovascular atherosclerosis), cerebrovascular disease, venous thrombosis, myocardial infarction, dyslipidemia, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis.

32. A method of reducing the level of apolipoprotein(a) in a subject, the method comprising the step of administering to the subject the dsRNA of any one of claims 1-24, the cell of claim 25, or the pharmaceutical composition of claim 26.

33. The method of any one of claims 28-32, wherein the dsRNA, cell, or pharmaceutical composition is administered subcutaneously, topically, or intravenously.

34. The method of any one of claims 28-33, wherein the subject is a human.

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