SiRNA for treating cardiovascular related diseases and conjugate and application thereof

Abstract

The invention provides siRNA for inhibiting LPA gene expression and a conjugate thereof, the siRNA comprises a positive-sense strand and an antisense strand, the positive-sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II; each nucleotide in the nucleotide sequence I and the nucleotide sequence II is modified or unmodified nucleotide; the nucleotide sequence I and the nucleotide sequence II are at least partially reversely complementary to form a double-stranded region; the nucleotide sequence I is basically consistent with a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence of which the length is at least 15 nucleotides in mRNA expressed by the LPA gene. The siRNA as well as the conjugate and the pharmaceutical composition thereof provided by the invention have strong inhibitory ability on LPA genes, can significantly reduce the expression quantity of LPA mRNA, and are low in drug toxicity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to siRNAs that inhibit LPA gene expression, their conjugates, and their applications. Background Technology

[0002] Atherosclerotic cardiovascular disease (ASCVD) is one of the greatest threats to global public health, with a high incidence and a trend towards affecting younger people. Dyslipidemia is a significant risk factor for ASCVD. The two most common lipid components in clinical practice are cholesterol and triglycerides. Cholesterol and triglycerides in the blood are primarily found in lipoproteins, including chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (MDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and lipoprotein(a) (Lp(a)).

[0003] Lp(a) carries a large amount of cholesterol and promotes atherosclerosis. It has also been proven to be an independent risk factor for cardiovascular diseases such as atherosclerosis and aortic stenosis. Lp(a) is an LDL-like particle, mainly produced in the liver, and has atherogenic, thrombogenic, and pro-inflammatory properties, potentially having pathological effects on multiple systems. Individual plasma Lp(a) levels are mainly determined by genetics and therefore remain relatively stable throughout life. Approximately 20% of the global population has Lp(a) >50 mg / dL. Studies have shown that for every 50 nM increase in Lp(a) content in apolipoprotein B, the risk of coronary heart disease increases by 28%, while for every 50 nM increase in LDL content in apolipoprotein B, the risk of coronary heart disease increases by only 4%. Studies have shown that even with effective control of LDL-C, Lp(a) increases the risk of cardiovascular events. Research indicates that elevated Lp(a) is associated with coronary heart disease, cerebrovascular disease, familial hypercholesterolemia, kidney disease, diabetes, hypothyroidism, and peripheral vascular disease.

[0004] Currently, there is a lack of effective treatments to lower Lp(a) levels. Statins have limited ability to lower Lp(a) and may even increase it. There are also no drugs approved specifically for lowering Lp(a). Niacin, PCSK9 inhibitors, cholesterol ester transporter inhibitors, and ApoB synthesis inhibitors can lower Lp(a) levels by 20%-30%, but there is no evidence of effective cardiovascular benefit.

[0005] Compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate the expression of upstream genes and are relatively less likely to cause drug resistance; moreover, small nucleic acid drugs have a long half-life in the body, so the frequency of administration is low (they can be given once every six months), and patients have good compliance. Summary of the Invention

[0006] The purpose of this invention is to provide an siRNA molecule that can inhibit LPA gene expression, in order to provide a new treatment for diseases related to atherosclerotic cardiovascular disease (ASCVD) caused by elevated Lp(a) levels.

[0007] In a first aspect, this disclosure provides an siRNA for inhibiting LPA gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, and the antisense strand comprising a nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence (difference of 0, 1, 2, or 3 nucleotides), the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressing the LPA gene, preferably, the first nucleotide sequence being a nucleotide sequence of 15 to 25 nucleotides in length in the mRNA expressing the LPA gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; Preferably, the mRNA expressing the LPA gene is as shown in NCBI refseqID NM_005577.4; specifically, the mRNA sequence is as shown in SEQ ID NO: 1.

[0008] In some implementations, the nucleotide sequence II in the above-described siRNA is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence.

[0009] In some embodiments, in any of the siRNAs described above, the sense and antisense strands may be of the same or different lengths, with the sense strand being 16-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. In some embodiments, the length ratio of the sense to antisense strands of the siRNA is 19 / 21, 21 / 23, or 19 / 24.

[0010] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the first nucleotide segment.

[0011] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence I comprises at least 15 consecutive nucleotides differing by 0, 1, 2 or 3 nucleotides from those in SEQ ID NO:2-116, such as at least 15, 16, 17, 18, 19, 20 or 21 nucleotides; preferably, the nucleotide sequence I is as shown in SEQ ID NO:2-116.

[0012] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence II comprises at least 15 consecutive nucleotides differing from those in SEQ ID NO:117-231 by 0, 1, 2, or 3 nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides; preferably, the nucleotide sequence II is as shown in SEQ ID NO: 117-231.

[0013] In some embodiments, in any of the siRNAs described above, the sense strand comprises the sense strand of any of the siRNAs shown in Table 1 of this document, and the antisense strand comprises the antisense strand of the corresponding siRNA in Table 1.

[0014] In some embodiments, the nucleotide sequence I in the siRNA is shown as SEQ ID NO: 3, 10, 13, 18, 20, 24, 69, 76, 90 and 99, respectively, and the nucleotide sequence II is shown as SEQ ID NO: 118, 125, 128, 133, 135, 139, 184, 191, 205 and 214, respectively.

[0015] In some embodiments, in any of the above-described siRNAs, the sense strand is of the same length as the sense strand of any of the siRNAs shown in Table 1 and differs by one nucleotide; preferably, the difference of one nucleotide is Z1 located at the 3' end, Z1 being optionally A, U, C or G; even more preferably, in any of the above-described siRNAs, the antisense strand has Z2 at the 5' end that is complementary to Z1.

[0016] In some embodiments, in any of the siRNAs described above, each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified nucleotide, wherein the modified nucleotide is a fluorinated nucleotide or a non-fluorinated nucleotide.

[0017] In some embodiments, the fluorinated nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, having the structure shown in formula (1); the non-fluorinated nucleotide refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group. These nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides or nucleotide analogs may be selected from 2'-alkoxy-modified nucleotides or nucleotide analogs, 2'-substituted alkoxy-modified nucleotides or nucleotide analogs, 2'-alkyl-modified nucleotides or nucleotide analogs, 2'-substituted alkyl-modified nucleotides or nucleotide analogs, 2'-amino-modified nucleotides or nucleotide analogs, 2'-substituted amino-modified nucleotides or nucleotide analogs, and 2'-deoxynucleotides. In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (2). In some embodiments, the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl-modified nucleotide (2'-MOE), as shown in formula (3). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (5).

[0018]

[0019] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine ribonucleotide. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleotides, or acyclic nucleotides.

[0020] A bridging nucleotide is a restricted or inaccessible nucleotide. Bridging nucleotides can contain a five-membered, six-membered, or seven-membered ring with a fixed C3'-endo sugar puckering. In some embodiments, the bridging nucleotide can be LNA, ENA, cET BNA, etc.; wherein LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8).

[0021]

[0022] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some implementations, acyclic nucleotides can be unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA), where UNA is shown in formula (9) and GNA is shown in formula (10).

[0023]

[0024] In formulas (9) and (10) above, R is selected from H, OH or alkoxy (O-alkyl).

[0025] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1' position to the 2' or 3' position on the ribose ring, as shown in formula (11) or formula (12).

[0026]

[0027] In formulas (11) and (12), R is selected from H, OH, F or non-fluorine groups as described above.

[0028] In equations (1) to (12), Base represents a base.

[0029] In some embodiments, in any of the above-described siRNAs, one or more nucleotides at positions 5, 7, 8, 9, 10, 11, and 12 of nucleotide sequence I are fluorinated nucleotides, in the direction from the 5' end to the 3' end; and one or more nucleotides at positions 2, 3, 5, 6, 7, 8, 9, 10, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, in the direction from the 5' end to the 3' end. Preferably, the nucleotides at positions 7, 9, 10, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, 10, 11, and 12 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, 8, 9, and 10 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides in the direction from the 5' end to the 3' end.

[0030] Preferably, the nucleotide sequence II is positioned at position 4, following a direction from the 5' end to the 3' end. Position 8 contains at least one (e.g., one, two, three, four, five or more) heat-stabilizing modification; preferably, position 6, 7 or 8 contains one heat-stabilizing modification; preferably, the heat-stabilizing modification may include, but is not limited to, debasement modification, mismatch with a relative nucleotide in the opposite chain, or sugar modification such as 2'. Deoxygenated or acyclic nucleotides, or those with impaired WC H pairs that are complementary to the target mRNA. The bonded nucleotides may be modified with phosphate esters.

[0031] In some embodiments, at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate ester groups with modifying groups. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom. In some embodiments, the phosphate ester group with modifying groups is a thiophosphate ester group having the structure shown in formula (13).

[0032]

[0033] In some embodiments, in any of the above-described siRNAs, the thiophosphate group linkage is present at least one of the following positions: between the first and second nucleotides of the sense and / or antisense strands; between the second and third nucleotides of the sense and / or antisense strands; between the 19th and 20th nucleotides of the antisense strand; between the 20th and 21st nucleotides of the antisense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof. In some embodiments, in any of the above-described siRNAs, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide, as shown in formulas (14), (15) and (16).

[0034]

[0035] In a second aspect, this disclosure provides an siRNA conjugate comprising any of the siRNAs described above and a conjugating group conjugated to the siRNA. In some embodiments, the pharmaceutically acceptable conjugating group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, or tetravalent. In some embodiments, the conjugation site between the siRNA and the conjugating group may be at the 3' or 5' end of the sense strand of the siRNA, at the 3' end of the antisense strand, or within the internal sequence of the siRNA.

[0036] In some embodiments, the conjugating group in the above siRNA conjugate is L96, with the structure shown below:

[0037] Formula (I).

[0038] The siRNA conjugate has its sense strand selected from the nucleotide sequence shown in the following formula: 5'-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXf ... XmXm-L96-3' or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3' or 5'-XmsXmsXmXmXmXfXmX fXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3' The antisense strand in the siRNA conjugate is selected from the nucleotide sequence shown in the following formula: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmfXmXfXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmfXmXfXmXmXmXmXmXmsXms-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXm-3', or 5'-VPXmsXfsXmXmXmXmXf ... mXfXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXf sXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXm XmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXfXmXfXmXfXmXmXm XmXfXmXfXfXmXmXfXmXfXmXfXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXm XmsXm - 3', or 5' - VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmsXm - 3', or 5' - VPXms mXfXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXfXmXfXmXfXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3'.

[0039] Wherein, Xm represents any nucleotide modified with 2'-methoxy, such as 2'-methoxy modified C, G, U, A, T; Xf represents any nucleotide modified with 2'-fluoride, such as 2'-fluoride modified C, G, U, A, T; lowercase s indicates that the two nucleotides adjacent to the letter s on the left and right are linked by thiophosphate subunits; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

[0040] In some implementations, the justice chain and the antisense chain are combined as follows: M-1: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmXmXmsXmsXm-3'; Or M-1': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm-3'; Or M-2: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXmsXm-3'; Or M-2': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3'; Or M-3: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXfXmXmXmXmXmXmXm-3'; or M-3' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXfXmXmXmXmXm-3'; Or M-4: Justice Chain: 5'-XmsXmsXmXmXmXmXfXmXfXmXmXfXmXmXmXmXmXmXmXmXm-L96-3'; Antisense Chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXfXmXmXmXmXmXmXmXmXmXm-3'; or M-4' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXfXmXmXfXmXmXfXmXmXmXmXmsXmsXm-3'; Or M-5: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXfXmXmXmXmXfXmXmXmXmXmXmXmXmXmXmsXmsXm-3'; Or M-5': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXmXfXmXmXmXmXmXmXmXmXmXmXmXmXmsXmsXm-3'; Or M-6: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3'; Or M-6': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXm-3'.

[0041] In some embodiments, the siRNA conjugate comprises a sense strand and an antisense strand, wherein the sense strand comprises the sense strand of any of the siRNA conjugates shown in Table 2 of this document, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate.

[0042] In some embodiments, the siRNA conjugate comprises a sense strand and an antisense strand, wherein the sense strand comprises the sense strands of YG-2M1, YG-9M1, YG-12M1, YG-17M1, YG-19M1, YG-23M1, YG-68M1, YG-75M1, YG-89M1, YG-98M1, YG-2M2, YG-12M2, YG-23M2, YG-89M2, YG-98M2, YG-89M3, YG-89M4, YG-9M5, YG-12M5, YG-89M5, and YG-89M6, the nucleic acid sequences of which are shown in SEQ ID. As shown in SEQ ID NOs 234, 235, 237, 238, 240, 242, 251, 252, 256, 259, 262, 263, 264, 265, 266, 268, 270, 271, 272, 273, and 275, the antisense strand comprises the antisense strand of the corresponding siRNA conjugate, and its nucleic acid sequences are shown in SEQ ID NOs 277, 278, 280, 281, 283, 285, 294, 295, 299, 302, 305, 306, 307, 308, 309, 311, 313, 314, 315, 316, and 318, respectively.

[0043] In a third aspect, this disclosure provides a composition comprising any of the siRNAs or siRNA conjugates described above.

[0044] In some embodiments, the composition is a pharmaceutical composition and further includes a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers or excipients involved in this disclosure include, but are not limited to, water for injection, sodium hydroxide, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, phosphoric acid, sodium chloride, potassium chloride, hydrochloric acid, anhydrous potassium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, PEG2000, PEG6000, cholesterol, distearate, glyceryl 1,2-dimyristate, and dimethyl adipic acid.

[0045] In a fourth aspect, this disclosure provides the use of any of the foregoing siRNAs, siRNA conjugates, or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of diseases associated with LPA expression.

[0046] In some implementations, the diseases associated with elevated Lp(a) particle levels include ASCVD; The diseases involved in ASCVD include, but are not limited to: coronary heart disease, cerebrovascular disease, familial hypercholesterolemia, kidney disease, diabetes, hypothyroidism, and peripheral vascular disease. Coronary heart disease includes chronic coronary artery disease, also known as chronic myocardial ischemia syndrome, which includes stable angina, ischemic cardiomyopathy, and occult coronary heart disease. Acute coronary syndrome includes unstable angina, non-ST-segment elevation myocardial infarction, and ST-segment elevation myocardial infarction.

[0047] Cerebrovascular diseases are broadly classified into two categories: ischemic cerebrovascular diseases and hemorrhagic cerebrovascular diseases. Ischemic cerebrovascular diseases include, but are not limited to, cerebral infarction and transient ischemic attacks. Hemorrhagic cerebrovascular diseases include, but are not limited to, cerebral hemorrhage and subarachnoid hemorrhage.

[0048] Kidney diseases include chronic kidney disease (CKD) or nephrotic syndrome.

[0049] Peripheral vascular diseases include, but are not limited to, lower extremity arteriosclerosis obliterans, vasculitis, arterial embolism, arterial thrombosis, venous thrombosis, carotid and vertebral artery stenosis, pulmonary embolism, renal artery stenosis, aortic dissection aneurysm, thoracic and abdominal aortic aneurysm, Budd-Chiari syndrome, etc.

[0050] The pharmaceutical compositions disclosed herein can be used alone or in combination with standard oral lipid-lowering drugs, providing trial support for diversified treatment options for clinical ASCVD patients.

[0051] Based on the amount of siRNA contained therein, the generally suitable dosage range of the siRNA, siRNA conjugate or pharmaceutical composition relating to the present disclosure that inhibits LPA gene expression will be from about 0.1 mg / kg to about 10.0 mg / kg, preferably from about 0.3 mg / kg to about 3.0 mg / kg.

[0052] The routes of administration disclosed herein include intravenous administration, subcutaneous administration, intrathecal injection, intramuscular administration, transdermal administration, airway administration (aerosol), ocular administration, nasal administration, rectal administration, pulmonary administration, and local administration (including oral administration and sublingual administration).

[0053] The siRNA, its conjugates, and pharmaceutical compositions disclosed herein can specifically target the liver, pair complementaryly with the liver LPA mRNA sequence, induce LPA mRNA degradation, thereby inhibiting the synthesis of LPA genes in the liver, resulting in a sustained reduction of Lp(a) protein, exerting a sustained Lp(a)-lowering effect, while avoiding off-target effects and exhibiting low cytotoxicity, and can fundamentally prevent or treat ASCVD caused by elevated Lp(a) levels.

[0054] In addition, compared with traditional small molecule drugs and antibody drugs, small nucleic acid drugs can directly regulate the expression of upstream genes and are relatively less likely to develop drug resistance; moreover, small nucleic acid drugs have a long half-life in the body, so the frequency of administration is low (they can be given once every six months), and patients have good compliance.

[0055] In summary, the siRNA, its conjugates, and pharmaceutical compositions disclosed herein exhibit strong inhibitory activity against the LPA gene, significantly reducing LPA mRNA expression levels, and possess low drug toxicity. Therefore, the siRNA, its conjugates, and pharmaceutical compositions disclosed herein can effectively prevent and / or treat diseases associated with LPA expression, providing patients with more effective, safe, and convenient therapeutic drugs, and have promising prospects as pharmaceutical products.

[0056] In another aspect of this disclosure, a modified siRNA or a conjugate thereof is provided, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I, and the antisense strand comprising nucleotide sequence II; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length from the mRNA expressing the target gene, characterized in that: In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, or the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides; and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions are 2' methylated nucleotides. Preferably, the antisense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, between the second and third bases, and between the first and second bases at the 3' end; the sense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, and between the second and third bases. Preferably, the 5' terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide; more preferably, it is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

[0057] Preferably, the siRNA is conjugate formed by linking one or more GalNAc derivatives through a divalent or trivalent branched structure; Preferably, the siRNA conjugate has the following structure: M-2: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXmsXm-3'; Or M-2': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3'.

[0058] In another aspect of this disclosure, a modified siRNA or a conjugate thereof is provided, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising nucleotide sequence I, and the antisense strand comprising nucleotide sequence II; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length from the mRNA expressing the target gene, characterized in that: In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, or the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'methoxy (2'-OMe) nucleotides. Preferably, the antisense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, between the second and third bases, and between the first and second bases at the 3' end; the sense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, and between the second and third bases. Preferably, the 5' terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog; Preferably, the siRNA is conjugate formed by linking one or more GalNAc derivatives through a divalent or trivalent branched structure; Preferably, the siRNA conjugate has the following structure: M-3: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3' Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXfXmXmXmXmXmXmXm-3'; or M-3' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXmXfXmXmXmXmsXmsXm-3'.

[0059] Another aspect of this disclosure provides a modified siRNA or a conjugate thereof, said siRNA comprising a sense strand and an antisense strand, said sense strand comprising nucleotide sequence I, said antisense strand comprising nucleotide sequence II; said nucleotide sequence I and said nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; said nucleotide sequence I is substantially identical to a first nucleotide sequence, said first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length from the mRNA expressing the target gene, characterized in that: In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, or the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'methoxy (2'-OMe) nucleotides. Preferably, the antisense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, between the second and third bases, and between the first and second bases at the 3' end; the sense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, and between the second and third bases. Preferably, the 5' terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog; Preferably, the siRNA is conjugate formed by linking one or more GalNAc derivatives through a divalent or trivalent branched structure; Preferably, the siRNA conjugate has the following structure: M-4: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3' Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXfXmXmXfXmXmXmXmXmXmXmsXmsXm-3'; or M-4' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3' Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXfXmXmXfXmXmXfXmXmXmXmsXmsXm-3'.

[0060] Another aspect of this disclosure provides a modified siRNA or a conjugate thereof, said siRNA comprising a sense strand and an antisense strand, said sense strand comprising nucleotide sequence I, said antisense strand comprising nucleotide sequence II; said nucleotide sequence I and said nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; said nucleotide sequence I is substantially identical to a first nucleotide sequence, said first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length from the mRNA expressing the target gene, characterized in that: In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, 11, and 12 of nucleotide sequence I are fluorinated nucleotides, or the nucleotides at positions 5, 7, 8, 9, and 10 of nucleotide sequence I are fluorinated nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 3, 5, 7, 10, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'methoxy (2'-OMe) modified nucleotides; Preferably, the antisense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, between the second and third bases, and between the first and second bases at the 3' end; the sense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, and between the second and third bases. Preferably, the 5' terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog; Preferably, the siRNA is conjugate formed by linking one or more GalNAc derivatives through a divalent or trivalent branched structure; Preferably, the siRNA conjugate has the following structure: M-5: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXfXmXmXmXmXfXmXmXmXmXmXmXmXmXmXmsXmsXm-3'; or M-5' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXfXmXfXmXfXmXmXmXfXmXmXmXmXmXmXmXmXmXmsXmsXm-3'.

[0061] Another aspect of this disclosure provides a modified siRNA or a conjugate thereof, said siRNA comprising a sense strand and an antisense strand, said sense strand comprising nucleotide sequence I, said antisense strand comprising nucleotide sequence II; said nucleotide sequence I and said nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; said nucleotide sequence I is substantially identical to a first nucleotide sequence, said first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length from the mRNA expressing the target gene, characterized in that: In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, or the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides; and in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, and the nucleotides at the remaining positions are 2'methoxy (2'-OMe) nucleotides. Preferably, the antisense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, between the second and third bases, and between the first and second bases at the 3' end; the sense strand of the siRNA contains phosphate thioester substitutions between the first and second bases at the 5' end, and between the second and third bases. Preferably, the 5' terminal nucleotide of the siRNA antisense strand is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog; Preferably, the siRNA is conjugate formed by linking one or more GalNAc derivatives through a divalent or trivalent branched structure; Preferably, the siRNA conjugate has the following structure: M-6: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3'; or M-6' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXm-3'.

[0062] Beneficial technical effects The siRNA, its conjugates, and pharmaceutical compositions disclosed herein exhibit strong inhibitory activity against the LPA gene, significantly reducing LPA mRNA expression levels, and possess low drug toxicity. Therefore, the siRNA, its conjugates, and pharmaceutical compositions disclosed herein can effectively prevent and / or treat diseases associated with LPA expression, providing patients with more effective, safe, and convenient therapeutic drugs, and have promising prospects as pharmaceutical products. Attached Figure Description

[0063] Figure 1 The results of the cytotoxicity experiments of the siRNA conjugate disclosed herein are as follows.

[0064] Figure 2 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in vivo.

[0065] Figure 3 This is a dose-dependent result of the siRNA conjugate disclosed herein inhibiting hLp(a) expression in vivo.

[0066] Figure 4 This is a dose-dependent result of the siRNA conjugate disclosed herein inhibiting hLp(a) expression in vivo.

[0067] Figure 5 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in mice.

[0068] Figure 6 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in mice.

[0069] Figure 7 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in mice.

[0070] Figure 8 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in mice.

[0071] Figure 9 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in mice.

[0072] Figure 10 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in mice.

[0073] Figure 11 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in mice.

[0074] Figure 12 The results show that the siRNA conjugate disclosed herein inhibits hLp(a) expression in cynomolgus monkeys. Detailed Implementation

[0075] I definition Unless otherwise specified, in the preceding and following text, uppercase letters C, G, U, and A represent cytosine, guanine, uracil, and adenine nucleotides, and uppercase letter X represents C, G, U, or A; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by a thiophosphate subunit; the letter combination VP indicates that the nucleotide adjacent to the right of letter combination VP is a vinylphosphonate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; L96 has the structure of formula (I) and is linked to the 3' end of the positive chain via a phosphate ester bond.

[0076] In the preceding and following text, "fluorinated nucleotides" refers to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, and "non-fluorinated nucleotides" refers to nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotides" refers to nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group.

[0077] As used herein, “includes” is intended to be used interchangeably with the phrase “includes but is not limited to”. The term “or” is used herein to mean “and / or” and is used interchangeably with that term unless the context clearly indicates otherwise.

[0078] The term "LPA" refers to a lipoprotein (a) encoding gene having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, humans, cattle, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs, unless otherwise stated. The term also refers to fragments or variants of natural LPA that retain at least one in vivo or in vitro activity of natural LPA. The term encompasses both the full-length, unprocessed precursor form of LPA and the mature form resulting from post-translational cleavage of the signal peptide. The sequence of the human LPA AmRNA transcript can be found, for example, NCBI accession number NM_005577.4 (SEQ ID NO:1). The predicted sequence of rhesus monkey LPA mRNA can be found, for example, NCBI accession number XM_028847001.1. The sequence of cynomolgus monkey LPA mRNA can be found, for example, NCBI accession number XM_065543183.1. Certain sequences disclosed herein exhibit cross-reactivity with cynomolgus monkey LPA sequences, thus supporting preclinical assessment of LPA gene silencing activity in non-human primates. Further examples of LPA mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0079] As used in this article, "LPA" also refers to a specific polypeptide expressed in cells through naturally occurring DNA sequence variations in the LPA gene, such as single nucleotide polymorphisms (SNPs) of the LPA gene. Many SNPs in the LPA gene have been identified and can be found, for example, in NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov / snp). Non-restricted instances of SNPs in the LPA gene include rs1177315054 or rs1009755406.

[0080] As used herein, "target sequence" means a continuous nucleotide sequence portion of an mRNA molecule formed during LPA gene transcription, including mRNA that is a primary transcription product of RNA processing. In one embodiment, the target portion of the sequence is at least long enough to serve as a matrix for iRNA-mediated cleavage of the nucleotide sequence portion of the mRNA molecule formed at or near the LPA gene transcription.

[0081] Target sequences can be approximately 9 to 36 nucleotides in length, such as approximately 15 to 30 nucleotides. For example, target sequences can range from approximately 15-30 nucleotides, including sequences like 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, and 19-23. 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide lengths. The ranges and intermediate values ​​of the lengths cited above are also intended to be part of this disclosure.

[0082] As used herein, the term "chain containing a sequence" refers to an oligonucleotide containing a single nucleotide chain described using the sequence mentioned in standard nucleotide nomenclature.

[0083] Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences.

[0084] Unless otherwise specified above and below, "substantially identical" means that there are no more than three base differences between the two nucleotide sequences involved, such as no more than one, two, or three; "truly identical" means that there are no more than one base difference between the two nucleotide sequences; and "completely identical" means that there are no base differences between the two nucleotide sequences. Preferably, the difference is located at the 3' end of the antisense strand or the 5' end of the sense strand.

[0085] The siRNA molecules used in the compositions and methods of this disclosure are double-stranded RNAs and are referred to herein as "siRNA molecules," "double-stranded RNA (dsRNA) molecules," "dsRNA agents," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, described as having "sense" and "antisense" orientations relative to a target RNA (i.e., an LPA gene). In some embodiments of this disclosure, the double-stranded RNA (dsRNA) induces the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0086] Typically, the majority of nucleotides in each strand of an siRNA molecule are ribonucleotides; however, as detailed herein, each or both of the two strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, an siRNA molecule may include chemically modified ribonucleotides; an siRNA molecule may include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide link, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses substitution, addition, or removal of, for example, a functional group or atom, from the internucleotide link, sugar moiety, or nucleobase. Modifications applicable to pharmaceuticals disclosed herein include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, an siRNA molecule may contain any such modification as used in siRNA-type molecules.

[0087] The double-stranded region can be of any length, allowing for the specific degradation of a desired target RNA via a RISC pathway, and the length can range from about 9 to 36 base pairs, for example, about 15-30 base pairs, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19. 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25 21-24, 21-23, or 21-22 base pairs. The ranges and lengths listed above, as well as intermediate values ​​of the lengths, are also conceived to be part of this disclosure.

[0088] In one embodiment, the siRNA molecule of this disclosure is dsRNA, each strand of which includes 19-23 nucleotides that interact with a target RNA sequence, such as an LPA target mRNA sequence, to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into the cell is cleaved into siRNA by a type III endonuclease known as Dicer. The siRNA is then bound to an RNA-induced silencing complex (RISC), in which one or more helicases unfold the siRNA double strand, thereby enabling complementary antisense strand-guided target recognition. Upon binding to a suitable target mRNA, one or more endonucleases in the RISC cleave the target, inducing silencing.

[0089] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of the siRNA molecule. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA may include an overhang having at least one nucleotide; alternatively, the overhang may contain at least two, three, four, five, or more nucleotides. The nucleotide overhang may include or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs may be located on the sense strand, the antisense strand, or any combination thereof. Additionally, one or more nucleotides of the overhang may be present at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA, such as the 3' end dTdT overhang of the antisense strand conventionally used in the art.

[0090] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at its 3'-end and / or 5'-end. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, at its 3'-end and / or 5'-end. In another embodiment, one or more nucleotides in the overhang are replaced with nucleoside phosphate thioesters.

[0091] In some embodiments, the protrusion at the sense strand or antisense strand, or both, may include an extension length longer than 10 nucleotides, such as 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides. In some embodiments, the extended protrusion is located on the sense strand of the duplex. In some embodiments, the extended protrusion is located at the 3' end of the sense strand of the duplex. In some embodiments, the extended protrusion is located at the 5' end of the sense strand of the duplex. In some embodiments, the extended protrusion is located on the antisense strand of the duplex. In some embodiments, the extended protrusion is located at the 3' end of the antisense strand of the duplex. In some embodiments, the extended protrusion is located at the 5' end of the antisense strand of the duplex. In some embodiments, one or more nucleotides in the extended protrusion are replaced with nucleoside phosphate thioesters.

[0092] As used herein, the terms “flush” or “flat-ended” mean that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., no nucleotide overhangs. One or both ends of a dsRNA can be flush. When both ends of a dsRNA are flat-ended, the dsRNA is called flat-ended. For clarity, a “flat-ended” dsRNA is a dsRNA that is flush at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0093] The term "antisense strand" or "guide strand" refers to a strand of dsRNA that includes a region substantially complementary to a target sequence (e.g., an LPA mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence as defined herein, such as an LPA nucleotide sequence). In cases where the complementary region is not perfectly complementary to the target sequence, mismatches may occur within the molecule or in terminal regions. Typically, the most tolerant mismatches are found in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends of siRNA.

[0094] As used in this paper, the term "sense chain" or "passenger chain" refers to a chain of dsRNAs that contains regions substantially complementary to the regions of the antisense chain as defined herein.

[0095] "Inhibition of LPA gene expression" includes any level of LPA gene inhibition, such as at least partial inhibition of LPA gene expression, like at least about 20% inhibition. In some embodiments, the inhibition is 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%.

[0096] LPA gene expression can be assessed based on the level of any variable associated with LPA gene expression, such as LPA mRNA level or Lp(a) level. The level of inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. This control level can be any type of control level used in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only control or inert agent control) subjects, cells, or samples.

[0097] "Therapeutic effective amount" or "prophylactic effective amount" also includes the amount of RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The siRNA used in the methods of this disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0098] The phrase “pharmaceutically acceptable” as used herein refers to 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.

[0099] II. The siRNA compounds disclosed herein This disclosure provides an siRNA for inhibiting LPA gene expression. The siRNA comprises a sense strand and an antisense strand. The sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II. Each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide. Nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. Nucleotide sequence I is substantially identical to a first nucleotide sequence, which is a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressing the LPA gene. Preferably, the first nucleotide sequence is a nucleotide sequence of 15 to 25 nucleotides in length in the mRNA expressing the LPA gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. Preferably, the mRNA expressing the LPA gene is as shown in NCBI refseqID NM_005577.4; specifically, the mRNA sequence is as shown in SEQ ID NO: 1.

[0100] In some implementations, the nucleotide sequence II in the above-described siRNA is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence.

[0101] In some embodiments, in any of the siRNAs described above, the sense and antisense strands may be of the same or different lengths, with the sense strand being 16-23 nucleotides in length and the antisense strand being 19-26 nucleotides in length. In some embodiments, the length ratio of the sense to antisense strands of the siRNA is 19 / 21, 21 / 23, or 19 / 24.

[0102] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with the first nucleotide segment.

[0103] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence I comprises at least 15 consecutive nucleotides as shown in SEQ ID NO:2-116, such as at least 15, 16, 17, 18, 19, 20, or 21 nucleotides; preferably, the nucleotide sequence I is as shown in SEQ ID NO:2-116.

[0104] In some embodiments, in any of the above-described siRNAs, the nucleotide sequence II comprises at least 15 consecutive nucleotides as in SEQ ID NO:117-231, such as at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides; preferably, the nucleotide sequence II is as shown in SEQ ID NO: 117-231.

[0105] In some embodiments, in any of the siRNAs described above, the sense strand comprises the sense strand of any of the siRNAs shown in Table 1 of this document, and the antisense strand comprises the antisense strand of the corresponding siRNA in Table 1.

[0106] III. Modified siRNA molecules of this disclosure In one embodiment, the siRNA molecule of this disclosure is unmodified and does not contain, for example, chemical modifications and / or conjugation known in the art and described herein. In another embodiment, the RNA of the siRNA of this disclosure, such as dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In some embodiments of this disclosure, substantially all nucleotides of the siRNA molecule of this disclosure are modified nucleotides. In other embodiments of this disclosure, all nucleotides of the siRNA molecule of this disclosure are modified nucleotides. The phrase "substantially all nucleotides are modified nucleotides" in the siRNA molecule of this disclosure means that it is largely, but not entirely, modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotide.

[0107] The nucleic acids characterized in this disclosure can be synthesized and / or modified using methods well established in the art. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inversion) or 3'-end modifications (conjugation, DNA nucleotides, inversion, etc.); base modifications, such as substitution with a stable base, destabilization of a base, or a base that pairs with an expanded library of compatibility, base removal (debasement nucleotide), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and / or backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of siRNA compounds used in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or without natural internucleotide bonds. RNAs with modified backbones include those without phosphorus atoms in their backbones, among others. For the purposes of this specification and as sometimes discussed in the art, modified RNAs without phosphorus atoms in their internucleotide backbones may also be considered oligonucleotides. In some embodiments, the modified siRNA will have a phosphorus atom in its internucleotide backbone.

[0108] Modified RNA backbones include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl and other alkylphosphonates, including 3'-alkylene phosphonates and chiral phosphonates, hypophosphonates, phosphoramidites, including 3'-aminophosphatides and aminoalkylphosphatides, thiocarbonylphosphatides, thiocarbonylalkylphosphonates, thiocarbonylalkyl phosphate triesters, and borophosphates with normal 3'-5' bonds, analogs of these esters linked at 2'-5', and those esters with reverse polarity, wherein adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are different salts, mixed salts, and free acid forms.

[0109] The modified RNA may also contain one or more substituted sugar moieties. The siRNA molecule characterized herein may include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and alkynyl groups. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, wherein n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following groups at the 2' position: C1 to C2. 10Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, aryl alkyl groups, O-alkylaryl or O-aryl alkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter gene groups, intercalating agents, groups for improving the pharmacokinetic properties of iRNA or groups for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), i.e., alkoxy-alkoxy groups. Another exemplary modification is 2'-dimethylaminoethoxy, i.e., the O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described below in this example; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH3)2. Further examples of modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (all three having R and S isomers); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide). Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluorinated (2'-F). Similar modifications can be made at other sites on the RNA of siRNA, especially at the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in 2'-5' linked dsRNA. siRNA can also have sugar mimics, such as replacing the cyclobutyl moiety of the pentofuranosyl sugar.

[0110] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetylaminohexanoyl)-4-hydroxyproline (Hyp-C6-NHAC), N-(hexanoyl-4-hydroxyproline (Hyp-C6), N-(acetyl-4-hydroxyproline (Hyp-NHAC), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, reverse dT (idT), and others. Disclosure of such modifications can be found in PCT Publication No. WO 2011 / 005861.

[0111] Numerous modification modifiers that enhance activity or stability and reduce off-target effects have been reported, and this disclosure may use optional modification modifiers disclosed in the prior art.

[0112] In some embodiments, the modification pattern of the siRNA disclosed herein can be found in CN110582283B, and the specific patterns include: The antisense strand in the siRNA molecule contains a dethermating modification of the double strand, and the dethermating modification is located at one of positions 5, 6, 7, or 8, counting from the 5' end of the antisense strand, and the antisense strand contains 2, 3, 4, 5, or 6 2'-fluorine modifications, wherein the sense strand optionally contains a ligand.

[0113] In some embodiments, the modification pattern of the modified siRNA disclosed herein can be found in CN118401665A: The antisense strand contains (i) 7 to 9 2'-fluorinated nucleotides and (ii) 12 to 14 2'-O-methylated nucleotides. For example, the antisense strand may contain 7 or more 2'-fluorinated nucleotides at positions 2-12, 14-16, and 18. Alternatively or additionally, the antisense strand may contain 2'-fluorinated nucleotides at positions 2 or 3 and 14 and optionally 18. In another example, the antisense strand may contain 12 or more 2'-O-methylated nucleotides at positions 1, 2-13, and 15-21. Alternatively or additionally, the antisense strand may contain 2'-O-methylated nucleotides at positions 1, 13, 17, and 19-21. In some cases, the antisense strand may contain a PS bond between positions 1 and 2, positions 2 and 3, positions 19 and 20, and / or positions 20 and 21.

[0114] The sense strand contains (i) 2 to 5 2'-fluorinated nucleotides and (ii) 11 to 16 2'-O-methylated nucleotides. For example, the sense strand may contain 2'-fluorinated nucleotides at two or more sites, positions 5 and 7-11. Alternatively or additionally, the sense strand contains 2'-fluorinated nucleotides at positions 5 and 7-10, or at positions 5, 7, 9, and 11. In another example, the sense strand may contain 2'-O-methylated nucleotides at 11 or more sites, positions 1-6, 8, and 10-19. Alternatively or additionally, the sense strand contains 2'-O-methylated nucleotides at positions 4, 6, and 11-19. In an example, the sense strand may further contain 2'-O-methylated nucleotides at positions 8 and 10. In other examples, one or more sites, positions 1-3, of the sense strand may contain 2'-O-methylated nucleotides. Alternatively, one or more of positions 1-3 in the sense strand may be unmodified. Alternatively or additionally, the sense strand may contain a 2'-deoxynucleotide, optionally located at position 9. In some cases, the sense strand may contain a PS bond between positions 1 and 2, positions 2 and 3, positions 4 and 5, positions 5 and 6, positions 17 and 18, and / or positions 18 and 19.

[0115] The disclosed siRNA may also contain nucleobase modifications or substitutions (often simply referred to as "bases" in the field). As used herein, "unmodified" or "native" nucleobases include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymidine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymidine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-Mitrol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine; 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deadenine and 7-deadenine; and 3-deadenine and 3-deadenine.

[0116] Other modifications to the siRNA molecule disclosed herein include 5' phosphate esters or 5' phosphate ester mimics, such as 5'-terminal phosphate esters or phosphate ester mimics on the antisense strand of an RNAi agent. Suitable phosphate ester mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are hereby incorporated by reference. More preferably is the vinylphosphonate (5'-(E)-vinylphosphonate, E-VP) modification disclosed in CN103154014 B, with the following specific structure:

[0117] in: Bx1 is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine; T2 is a thiophosphate nucleotide linker group that connects a compound of formula IIe to an oligonucleotide chain; and G is a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2 or OCH2-N(H)-C(=NH)NH2.

[0118] In some embodiments, the fluorinated nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with fluorine, having the structure shown in formula (1); the non-fluorinated nucleotide refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group. These nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides or nucleotide analogs may be selected from 2'-alkoxy-modified nucleotides or nucleotide analogs, 2'-substituted alkoxy-modified nucleotides or nucleotide analogs, 2'-alkyl-modified nucleotides or nucleotide analogs, 2'-substituted alkyl-modified nucleotides or nucleotide analogs, 2'-amino-modified nucleotides or nucleotide analogs, 2'-substituted amino-modified nucleotides or nucleotide analogs, and 2'-deoxynucleotides. In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (2). In some embodiments, the 2'-substituted alkoxy-modified nucleotide may be, for example, a 2'-O-methoxyethyl-modified nucleotide (2'-MOE), as shown in formula (3). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is shown in formula (4). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (5).

[0119]

[0120] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine ribonucleotide. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleotides, or acyclic nucleotides.

[0121] A bridging nucleotide is a restricted or inaccessible nucleotide. Bridging nucleotides can contain a five-membered, six-membered, or seven-membered ring with a fixed C3-endoglucan condensation. In some embodiments, the bridging nucleotide can be LNA, ENA, cET BNA, etc.; wherein LNA is shown in formula (6), ENA is shown in formula (7), and cET BNA is shown in formula (8).

[0122]

[0123] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some implementations, acyclic nucleotides can be unlocked nucleic acids (UNA) or glycerol nucleic acids (GNA), where UNA is shown in formula (9) and GNA is shown in formula (10).

[0124]

[0125] In formulas (9) and (10) above, R is selected from H, OH or alkoxy (O-alkyl).

[0126] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1' position to the 2' or 3' position on the ribose ring, as shown in formula (11) or formula (12).

[0127]

[0128] In formulas (11) and (12), R is selected from H, OH, F or non-fluorine groups as described above.

[0129] In equations (1) to (12), Base represents a base.

[0130] In another embodiment of this disclosure, the dethermating modification is incorporated into the seed region of the antisense chain (i.e., at the 5' end of the antisense chain). End position 2 9) To reduce or suppress off-target gene silencing, thereby optimizing dsRNA molecules for RNA interference. In some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) dethermating modification of the duplex within the first nine nucleotide positions of the 5' region of the antisense strand. In some embodiments, the dethermating modification of the duplex is located at position 2. 9. Or preferably at position 4 8 (from the 5' of the antisense chain) (Starting from the end). In some other embodiments, the thermal stabilization modification of the duplex is located at position 6, 7, or 8 (from the 5' end of the antisense chain). (Starting from the end). In some other embodiments, the dethermating modification of the duplex is located at position 7 (from the 5' end of the antisense chain). (Starting from the end). The term "one or more dethermating modifications" includes one or more modifications of dsRNA that result in a lower total melting temperature (Tm) (preferably one, two, three, or four degrees lower than the Tm of dsRNA without such one or more modifications). In some embodiments, the dethermating modification of the duplex is located at position 2, 3, 4, 5, or 9 (from the 5' end of the antisense strand). (Starting from the end).

[0131] De-stabilizing modifications can include, but are not limited to, base removal modifications; mismatches with relative nucleotides in the opposite chain; and sugar modifications, such as 2'-nucleotides. Deoxygenation modifications or noncyclic nucleotides, such as unlocked nucleic acids (UNA) or glycol nucleic acids (GNA). Dethermating modifications of the duplex can also be mismatches (i.e., non-complementary base pairs) between the dethermating nucleotide and the corresponding nucleotide in the opposite strand of the dsRNA duplex. Furthermore, dethermating modifications of the duplex in the seed region of the antisense strand include impaired WC-H pairs with complementary bases on the target mRNA. Bonded nucleotides. Further examples of debased nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876 (which is incorporated herein by reference in its entirety). Dethermating modifications may also include universal bases whose ability to form hydrogen bonds with relative bases is reduced or eliminated, as well as phosphate ester modifications. Specific examples of these modifications can be found in CN110582283B.

[0132] IV. siRNA molecules conjugated to ligands The nucleic acid disclosed herein can be conjugated to a target ligand to form a conjugate.

[0133] This disclosure further provides a conjugate for inhibiting the expression of a target gene in a cell, the conjugate comprising a nucleic acid moiety and a ligand moiety, the nucleic acid moiety comprising nucleic acids as defined anywhere herein.

[0134] In the conjugates disclosed herein, the positive strand of the nucleic acid can be conjugated to the ligand moiety.

[0135] In the conjugates disclosed herein, the ligand portion may comprise one or more GalNAc ligands and their derivatives, such as a GalNAc portion or several GalNAc portions comprising the 5' end of the second strand of the nucleic acid.

[0136] Some ligands can possess endosomolytic properties. Endosomolytic ligands promote the lysis of endosomes and / or the transport of the compositions or components of this disclosure from endosomes to the cytoplasm. Endosomolytic ligands can be polyanionic peptides or peptide mimics that exhibit pH-dependent membrane activity and fusogenicity. Endosomolytic components may contain chemical groups that undergo charge changes or protonation in response to pH changes. Endosomolytic components can be linear or branched.

[0137] The ligand can be coupled to the nucleic acid at the 3' end, 5' end, and / or internal location. Preferably, the ligand is coupled to the nucleic acid via an insert tether or a linker.

[0138] In some embodiments, the nucleic acid is a double-stranded nucleic acid. In a double-stranded nucleic acid, a ligand may attach to one or both strands. In some embodiments, the double-stranded nucleic acid contains a ligand conjugated to the sense strand. In other embodiments, the double-stranded nucleic acid contains a ligand conjugated to the antisense strand.

[0139] Ligands can conjugate to nucleoside bases, sugar moieties, or internucleotide bonds in nucleic acid molecules. Conjugation with purine nucleoside bases or their derivatives can occur at any position, including within and outside the ring. Conjugation with pyrimidine nucleotides or their derivatives can also occur at any position. Conjugation with the sugar moieties of nucleosides can occur at any carbon atom. Conjugation with internucleotide bonds can occur at phosphorus atoms containing phosphorus bonds, or at oxygen, nitrogen, or sulfur atoms bonded to phosphorus atoms. For internucleotide bonds containing amines or amides, conjugation can occur at the nitrogen atom of the amine or amide or at a nearby carbon atom.

[0140] Ligands are typically carbohydrates, such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or polysaccharides. Ligands can attach to nucleic acids via linker sites. Linker sites can be monovalent, divalent, or trivalent linkers.

[0141] The efficient delivery of oligonucleotides, particularly the double-stranded nucleic acids of this disclosure, into cells in vivo is important and requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One method to achieve specific targeting is to conjugate a targeting moiety or ligand to the nucleic acid. The targeting moiety helps direct the nucleic acid to the desired target site, and for the desired receptor site, an appropriate targeting moiety needs to be conjugated so that the conjugated molecule can be absorbed by the target cell, such as through endocytosis. The targeting moiety or ligand can be any part or ligand capable of targeting a specific receptor.

[0142] For example, the asialic acid glycoprotein receptor (ASGP-R) is a high-capacity receptor that is highly abundant on hepatocytes. One of the first disclosures of trianthopteric glycosides is in US Patent No. 5,885,968. Conjugates having three GalNAc ligands and containing phosphate ester groups are known and described in Dubber et al. (2003). ASGP-R exhibits an affinity for N-acetyl-D-galactosylamine (GalNAc) that is 50 times greater than that for D-Gal.

[0143] Hepatocytes expressing lectins (asialic acid glycoprotein receptor; ASGPR) can be used to target drugs to the liver via covalent coupling of pharmaceutical substances with galactose or galactosamine, wherein the lectins specifically recognize the terminal β-galactosyl subunit of glycosylated proteins or other oligosaccharides. Furthermore, the multivalent effect achieved through repetition of the targeting unit can significantly enhance binding affinity.

[0144] ASGPR is a medium for active endosome transport of glycoproteins containing terminal β-galactosyl groups. Therefore, ASGPR is highly suitable for the targeted delivery of drug candidates such as nucleic acids, which must be delivered into cells.

[0145] Sugars (which can also be called ligands) can be selected to have affinity for at least one type of receptor on target cells. Specifically, the receptor is on the surface of mammalian hepatocytes, for example, the hepatic asialic acid glycoprotein receptor (ASGP-R).

[0146] The sugar can be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine, and fucose. The sugar can be N-acetylgalactosamine (GalNAc).

[0147] Therefore, the ligands used in this disclosure may comprise: (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker that conjugates the GalNAc moieties to a nucleic acid or sequence as defined in any of the preceding aspects. The linker may be a monovalent, divalent, trivalent, or tetravalent branched structure. Nucleotides may be modified as defined herein.

[0148] "GalNAc" represents 2-(acetylamino)-2-deoxy-D-pyranose, commonly referred to in the literature as N-acetylgalactosamine. References to "GalNAc" or "N-acetylgalactosamine" include both the β-form: 2-(acetylamino)-2-deoxy-β-D-pyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-pyranose. The β-form: 2-(acetylamino)-2-deoxy-β-D-pyranose and the α-form: 2-(acetylamino)-2-deoxy-α-D-pyranose are used interchangeably. Preferably, the compounds disclosed herein comprise the β-form, 2-(acetylamino)-2-deoxy-β-D-pyranose.

[0149] V. Pharmaceutical compositions disclosed herein This disclosure also includes pharmaceutical compositions and formulations comprising the siRNA of this disclosure. In one embodiment, a pharmaceutical composition comprising the siRNA as described herein and a pharmaceutically acceptable carrier is provided herein. Pharmaceutical compositions containing siRNA are suitable for treating diseases or disorders associated with the expression or activity of the LPA gene, such as hyperlipidemia (e.g., hyperlipidemia), dyslipidemia (e.g., atherogenic dyslipidemia, diabetic dyslipidemia, or mixed dyslipidemia), hyperlipoproteinemia, hyperapolipoprotein β-lipoproteinemia, coronary artery disease, myocardial infarction, peripheral artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic calcification, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, restenosis, renal artery stenosis, angina pectoris, cerebral arteriosclerosis, cerebrovascular disease, and venous thrombosis. In some embodiments, the siRNA described herein is used to treat subjects suffering from chronic heart disease (CHD) or any symptoms or conditions associated with cardiovascular disease (CVD). In some embodiments, the siRNA described in this disclosure is used to treat patients with hypercholesterolemia (e.g., statin-resistant hypercholesterolemia, and heterozygous or homozygous familial hypercholesterolemia) who have myocardial infarction (MI), peripheral artery disease (PAD), calcified aortic valve disease (CAVD), atherosclerotic cardiovascular disease (ASCVD), atherosclerosis, dyslipidemia, thrombosis, or stroke.

[0150] This pharmaceutical composition is formulated based on the delivery mode. Examples include compositions formulated for systemic administration via parenteral delivery, such as intravenous (IV) or subcutaneous delivery. Another example is compositions formulated for direct delivery to the liver, such as via infusion into the liver, such as via a continuous pump.

[0151] The pharmaceutical composition disclosed herein can be administered at a dose sufficient to inhibit LPA gene expression. Typically, a suitable dose of the siRNA disclosed herein will be in the range of about 0.001 to about 200.0 mg / kg body weight per day, generally in the range of about 1 mg to 50 mg / kg body weight per day. Typically, a suitable dose of the siRNA disclosed herein will be in the range of about 0.1 mg / kg to about 5.0 mg / kg, preferably about 0.3 mg / kg to about 3.0 mg / kg.

[0152] Repeated dosing regimens may include therapeutic doses of siRNA administered on top of routine dosing, such as from once every 1 day to once a year. In some embodiments, siRNA is administered approximately once a month to approximately once a quarter (i.e., approximately once every 3 months).

[0153] VI. Use of the pharmaceutical compositions disclosed herein This disclosure also provides the use of the disclosed siRNA and / or compositions containing the disclosed siRNA to prepare pharmaceutical compositions that reduce and / or inhibit LPA expression in cells. This use involves contacting cells with dsRNA and maintaining the cells for a sufficient period of time to achieve degradation of the mRNA transcript of the LPA gene, thereby inhibiting LPA gene expression in cells. The reduction in gene expression can be assessed by any method known in the art. For example, the reduction in LPA expression can be assessed by: measuring the mRNA expression level of LPA using methods conventional to those skilled in the art, such as the Northern ink spot assay or qRT-PCR; or measuring the protein value of apolipoprotein A (Lp(a)) using methods conventional to those skilled in the art, such as the Western ink spot assay or immunoassay, thereby assessing the reduction in LPA expression.

[0154] The use of the pharmaceutical compositions disclosed herein may include contact with cells in vitro or in vivo, i.e., the cells may be in the body of a subject.

[0155] Cells suitable for use in the treatment of patients according to this disclosure can be any cell expressing the LPA gene. Cells suitable for use in the treatment of patients according to this disclosure can be mammalian cells, such as primate cells (e.g., human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (e.g., cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell, such as a human liver cell.

[0156] LPA expression in cells was inhibited by at least approximately 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%. %, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%. In a preferred embodiment, at least 20% of LPA expression is suppressed.

[0157] In vivo use of this disclosure may include administering a composition containing siRNA to a subject, wherein the siRNA comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the mammalian LPA gene to be treated. When the organism to be treated is a mammal (e.g., a human), the composition may be administered by any means known in the art, including but not limited to oral, intraperitoneal, or parenteral routes (including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, percutaneous, airway (aerosol), nasal, rectal, and local (including oral and sublingual) administration). In some embodiments, these compositions are administered by intravenous infusion or injection. In some embodiments, the compositions are administered by subcutaneous injection.

[0158] In some embodiments, administration is via a long-acting injection. Accumulated injection can release the siRNA in a coherent manner over an extended period. Therefore, accumulated injection can reduce the frequency of administration required to achieve the desired effect, such as a desired inhibitory, therapeutic, or prophylactic effect on LPA. Accumulated injection can also provide more consistent serum concentrations. Accumulated injection includes subcutaneous or intramuscular injection. In a preferred embodiment, the accumulated injection is a subcutaneous injection.

[0159] The administration mode can be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration can be selected for enhanced targeting.

[0160] In one aspect, this disclosure also provides the use of the aforementioned pharmaceutical composition to inhibit LPA gene expression in mammals. This use comprises administering a composition to mammals, including dsRNA targeting the LPA gene in mammalian cells, and maintaining the mammalian cells for a sufficient period of time to achieve degradation of the LPA gene mRNA transcript, thereby inhibiting LPA gene expression in the cells. The reduction in gene expression can be evaluated by any method known in the art or by the methods described herein (e.g., qRT-PCR). The reduction in protein products can be assessed by any method known in the art or by the methods described herein (e.g., ELISA). In embodiments, liver biopsy is used as tissue material to monitor the reduction in LPA gene and / or protein expression.

[0161] The disclosed siRNA can be administered as “free siRNA”. Free siRNA is administered in the absence of the pharmaceutical composition. Naked siRNA can be in a suitable buffer solution. This buffer solution may contain acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and molar osmotic concentration of the buffer solution containing the siRNA can be adjusted to make it suitable for administration to a subject.

[0162] Alternatively, the siRNA disclosed herein may be administered as a pharmaceutical composition, such as siRNA liposome formulations.

[0163] Subjects who may benefit from reduced and / or inhibited LPA gene expression include those with coronary artery disease, cerebrovascular disease, familial hypercholesterolemia, kidney disease, diabetes, hypothyroidism, and peripheral vascular disease. Coronary artery disease includes chronic coronary artery disease (also known as chronic myocardial ischemia syndrome), including stable angina, ischemic cardiomyopathy, and occult coronary artery disease; acute coronary syndrome includes unstable angina, non-ST-segment elevation myocardial infarction, and ST-segment elevation myocardial infarction. Cerebrovascular disease includes two main categories: ischemic cerebrovascular disease and hemorrhagic cerebrovascular disease. Ischemic cerebrovascular disease includes, but is not limited to, cerebral infarction and transient ischemic attack. Hemorrhagic cerebrovascular disease includes, but is not limited to, cerebral hemorrhage and subarachnoid hemorrhage. Kidney disease includes chronic kidney disease (CKD) or nephrotic syndrome. Subjects with peripheral vascular diseases include, but are not limited to, lower extremity arteriosclerosis obliterans, vasculitis, arterial embolism, arterial thrombosis, venous thrombosis, carotid and vertebral artery stenosis, pulmonary embolism, renal artery stenosis, aortic dissection aneurysm, thoracic and abdominal aortic aneurysm, Budd-Chiari syndrome, etc.

[0164] Administration of the siRNA according to this disclosure can result in a reduction in the severity, signs, symptoms, and / or markers of such disease or disorder in patients with dyslipidemia. In this context, "reduction" means a statistically significant reduction at this level. This reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0165] The efficacy of a pharmaceutical composition for the treatment or prevention of a disease can be evaluated, for example, by measuring the level of disease progression, disease mitigation, symptom severity, pain reduction, quality of life, the dosage of the agent required to maintain the therapeutic effect, disease biomarkers, or any other measurable parameter applicable to the treatment or targeted for prevention of a given disease. Monitoring the efficacy of treatment or prevention by measuring any one or any combination of such parameters is within the capabilities of those skilled in the art. For example, the efficacy of treatment for dyslipidemia can be assessed by, for instance, periodically monitoring one or more serum lipid values. Comparison of later data with initial data provides a physician with an indication of whether the treatment is effective. Monitoring the efficacy of treatment or prevention by measuring any one or any combination of such parameters is within the capabilities of those skilled in the art. In relation to the administration of siRNA or pharmaceutical compositions targeting LPA, “effective resistance” to dyslipidemia indicates that administration in a clinically appropriate manner results in a beneficial effect on at least a statistically significant portion of patients, such as improvement of symptoms, cure, reduction of disease, extended lifespan, improved quality of life, or effects generally recognized as positive by a physician familiar with the treatment of dyslipidemia and its related causes.

[0166] A therapeutic or preventative effect is evident when there is a statistically significant improvement in one or more parameters of a disease state, or when symptoms that could otherwise be expected cease to worsen or progress. As an example, a favorable change of at least 10%, and preferably at least 20%, 30%, 40%, 50%, or more, in a measurable parameter of the disease can indicate effective treatment. The efficacy of a given siRNA drug or formulation thereof can also be determined using experimental animal models of a given disease, as known in the art. When using experimental animal models, therapeutic efficacy is evident when a statistically significant reduction in biomarkers or symptoms is observed.

[0167] Before administering the full dose of siRNA, a smaller dose, such as 5% infusion response, can be given to the patient, and adverse effects, such as allergic reactions, can be monitored. In another instance, the patient is monitored for unwanted immune stimulation, such as increased cytokine levels (e.g., TNF-α or INF-α).

[0168] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains. While similar or equivalent methods and materials may be used to implement or test the siRNA and methods embodied in this disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references submitted herein are incorporated herein by reference in their entirety. In case of conflict, this disclosure, including definitions, shall prevail. Furthermore, the materials, methods, and examples described are illustrative only and are not intended to be limiting.

[0169] Example Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0170] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0171] Example 1. siRNA Design and Synthesis 1.1 siRNA Design A set of siRNAs targeting the human LPA gene (human LPA gene: NCBI refseq ID NM_005577.4; NCBI Gene ID: 4018) was designed online using OligoWalk. The human NM_005577.4 REFSEQ mRNA is 6431 bases in length. Sequences similar to the human gene were excluded to avoid any toxicity.

[0172] 1.2 siRNA sequence synthesis siRNA was synthesized according to a standard oligonucleotide solid-phase synthesis protocol, including a negative control siRNA (siCtrl).

[0173] Oligonucleotide solid-phase synthesis protocol: Commercially available 5'-DMT-2'-TBDMS-rU phosphoramide monomers, 5'-DMT-2'-TBDMS-rA(Bz) phosphoramide monomers, 5'-DMT-2'-TBDMS-rC(Ac) phosphoramide monomers, and 5'-DMT-2'-TBDMS-rG(iBu) phosphoramide monomers were used. RNA was synthesized at a synthesis scale of 500 nM. A phosphoramide solution was prepared at a concentration of 50 mM, and 0.3 M benzylthiotetrazole (BTT) acetonitrile solution was used as an activator. During synthesis, a 0.1 M oxidizing agent (pyridine:THF:water = 20:78:2) was used to convert trivalent phosphorus to pentavalent phosphorus to stabilize the phosphate backbone. After synthesis, the sequence was ammonolyzed from the solid support and precipitated. The 2'-2'-O-tert-butyldimethylsilyl protecting group was removed with triethylamine trihydrofluoric acid.

[0174] For the synthesized RNA sequence, ammonolysis was performed at 55°C for 40 minutes using an ammonia:methylamine ratio of 1:1. After ammonolysis, the solid support CPG powder was removed, and the supernatant was dried. A protecting group remover was added, and the reaction was carried out at 60°C for 2 hours. Then, n-butanol was added at a 1:5 ratio, and the mixture was allowed to stand at -20°C for 30 minutes. The precipitate was collected by centrifugation. The precipitate was dissolved in RNase-free water and purified using reversed-phase chromatography (0.1M triethylamineacetic acid (TEAA) and acetonitrile). The purified sample was desalted by ultrafiltration with PBS and annealed to obtain siRNA. Verification of the obtained siRNA confirmed successful preparation of the target siRNA.

[0175] 1.3 siRNA sequence modification and conjugate synthesis Modified siRNAs are synthesized according to oligonucleotide solid-phase synthesis schemes. The modified nucleotide groups can be introduced into the siRNAs disclosed herein using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications are well known to those skilled in the art. L96 is conjugated to siRNA to synthesize siRNA conjugates, referring to the synthetic methods disclosed in WO2014025805A1 or WO2017015109A1.

[0176] The structure of the conjugation group L96 is shown below:

[0177] Formula (I).

[0178] Annealing of oligonucleotides to generate siRNA conjugates: The RNA oligomers to be annealed were prepared into a 200 μM solution using sterile RNase-free H2O (without RNase hydrolase). The annealing reaction system was set up as follows: 100 μL of the above solution (double-strand concentration of 10 nM) was placed in a 95°C water bath for 10 minutes (≥100 nM requires 20 minutes at high temperature) → immediately cooled in a 60°C water bath → the annealed solution was stored at 4°C. Equimolar amounts of complementary RNA solutions were then combined. The siRNA conjugate construction was confirmed to be correct.

[0179] The siRNA and siRNA conjugate solution were prepared into a dry powder for later use.

[0180] The sequences of the synthesized siRNA molecules are shown in Table 1 below: Table 1. siRNA sequence listing targeting LPA

[0181]

[0182]

[0183] The sequences of the synthesized siRNA conjugates are shown in Table 2 below: Table 2. Sequence listing of siRNA conjugates targeting LPA

[0184]

[0185]

[0186] In this context, uppercase letters C, G, U, A, and T represent cytosine, guanine, uracil, adenine nucleotide, and thymine deoxynucleotide, respectively; lowercase letter m indicates that the nucleotide adjacent to the left of m is methoxy-modified; lowercase letter f indicates that the nucleotide adjacent to the left of f is fluorinated; lowercase letter s indicates that the two nucleotides adjacent to s are linked by a thiophosphate subunit; VP indicates that the nucleotide adjacent to the right of VP is a vinylphosphonate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide; L96 has the structure of formula (I) and is linked to the 3' end of the positive chain via a phosphate ester bond; dT represents 2'-deoxythymine nucleotide.

[0187] Example 2. In vitro activity screening of siRNA in HCT116 cell line 2.1 Experimental Procedure 2.1.1 Cell Culture HCT116 cells (BNCC, BNCC287750) were cultured in DMEM complete medium (Eallbio, with 10% FBS added) at 37°C and 5% CO2. When the confluence reached 80%-90%, the cells were digested with trypsin, counted, and transfected.

[0188] 2.1.2 Preparation of siRNA dilution buffer (1) The dry powder of the siRNA to be tested was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0189] (2) Prepare 1000 nM siRNA dilution solution Z.

[0190] a) Take 50 μl of the 100 μM siRNA stock solution obtained in step (1) above, add 50 μl of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 50 μM. b) Take 2 μl of the 50 μM siRNA dilution solution obtained in step a) and add 18 μl of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 5 μM. c) Take 3 μl of the prepared siRNA stock solution X and add 12 μl of Opti-medium to obtain a 1000 nM siRNA dilution solution Z.

[0191] 2.1.3 HCT116 cell transfection Take 0.6 μl of Lipo® RNAiMAX transfection reagent (Thermo Fisher, catalog number: 13778150) and add 10 μl of Opti-medium to obtain Lipo® RNAiMAX transfection reagent dilution. Mix the Lipo® RNAiMAX transfection reagent dilution with the 1000 nM siRNA dilution Z prepared in step 2.1.2 at a volume ratio of 1:1 to prepare a transfection mixture. Let it stand for 5 minutes, take 10 μl of the transfection mixture and add it to a 96-well plate, and add 90 μl of HCT116 cells cultured in step 2.1.1 (final volume 100 μl / well, cell number 20,000 / well, siRNA concentration in this system is 50 nM). Incubate for 24 hours after transfection.

[0192] 2.1.4 RNA Extraction According to the instructions for the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: CL132-01), total RNA was extracted from HCT116 cells obtained in step 2.1.3.

[0193] 2.1.5 Quantitative Real-Time PCR The extracted total RNA was analyzed by reverse transcription and real-time PCR using the FlysisAmp Cells-to-CT 1-Step SYBR Green Kit.

[0194] 2.1.6 Results Analysis (1) Use the software of the 7500 Real-Time PCR instrument (Thermo Fisher) to automatically calculate the Ct value; (2) Calculate the relative expression level of the gene using the following formula: ΔCt1=Ct(LPA group)–Ct(ACTIN of LPA group) ΔCt2=Ct(siCtrl group)–Ct(siCtrl group's ACTIN) ΔCt = ΔCt1 - ΔCt2, where the siCtrl group is the negative control group; mRNA expression relative to the siCtrl group = 2 -ΔΔCt Inhibition rate (%) = (1) (mRNA expression relative to the siCtrl group) × 100%.

[0195] 2.2 Experimental Results The inhibitory effects of the siRNA of this invention are shown in Table 3 below: Table 3. Results of in vitro screening of siRNA in HCT116 cell line

[0196]

[0197]

[0198] The results showed that some of the siRNAs disclosed in this study could significantly inhibit the expression of the LPA gene in HCT116 cells at a concentration of 50 nM.

[0199] Example 3. Cytotoxicity of siRNA conjugates 3.1 Experimental Procedure 3.1.1 Cell Culture HCT116 cells were cultured in MEM complete medium (Eallbio, with 10% FBS added) at 37°C and 5% CO2. When the confluence reached 80%-90%, the cells were digested with trypsin, counted, and transfected.

[0200] 3.1.2 HCT116 cell transfection HCT116 cells were transfected using a method similar to that in Example 2, with 15,000 HCT116 cells seeded per well. After 72 hours of culture, the cytotoxicity of each siRNA conjugate was measured by determining the cell viability / cytotoxicity ratio in each sample, with the transfected siRNA conjugate concentrations being 50 nM, 5 nM, and 0.5 nM, respectively. Cell viability was measured by determining intracellular ATP content using a CellTiter-Glo (Promega, catalog number G7570) assay, according to the manufacturer's protocol. ToxiLight was used according to the manufacturer's protocol. TM (Lonza, catalog number LT07-217) Measures cytotoxicity in the supernatant.

[0201] 3.2 Experimental Results The cytotoxicity results of the siRNA conjugates are shown in [link to results]. Figure 1 The results showed that some of the siRNA conjugates disclosed in this study exhibited low cytotoxicity and good cell compatibility.

[0202] Example 4. In vivo activity screening of siRNA conjugates 4.1 Experimental Procedure Six- to eight-week-old male mice (C57BL / 6), weighing approximately 20g, were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. At least 14 days prior to drug administration, 25μg of a plasmid containing the human LPA mRNA sequence (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was injected via high-pressure injection into the tail vein over 6-8 seconds, with an injection volume of 8% of the mouse's body weight. The LPA-HDI transgenic mouse model was established 14 days after injection. Then, the mice were administered the drug subcutaneously at a dose of 3 mg / kg per mouse, with three mice per group. Liver samples were collected on day 9 (D9) after drug administration for mRNA expression detection. Total RNA was extracted using the Trizol method. Reverse transcription of mRNA was performed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Catalog No.: R323-01, Vazyme). Real-time quantitative PCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit (Catalog No.: Q711-03, Vazyme).

[0203] 4.2 Experimental Results The in vivo activity screening results of siRNA conjugates are shown in the figure. Figure 2 The results showed that some chemically modified siRNA sequences had good activity in mice and could significantly inhibit LPA gene expression.

[0204] Example 5. In vivo activity screening of siRNA conjugates 5.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered the conjugate at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and seven days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0205] 5.2 Experimental Results The in vivo activity screening results of different doses of siRNA conjugates are shown in the figure. Figure 3 The results showed that YG-2M1, YG-12M1, YG-23M1, YG-89M1, and YG-98M1 could significantly reduce the expression level of serum hLp(a) in mice in a dose-dependent manner.

[0206] Example 6. In vivo activity screening of siRNA conjugates 6.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered the conjugate at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and seven days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0207] 6.2 Experimental Results The in vivo activity screening results of different doses of siRNA conjugates are shown in the figure. Figure 4 YG-2M2, YG-12M2, YG-23M2, YG-89M2, and YG-98M2 can all significantly reduce the expression level of serum hLp(a) in mice in a dose-dependent manner.

[0208] Example 7. In vivo activity screening of siRNA conjugates 7.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered the conjugate at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and at 7, 14, 28, and 35 days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0209] 7.2 Experimental Results The in vivo activity screening results of different siRNA conjugates are shown in the figure. Figure 5 , Figure 5 A represents the result on day 7. Figure 5 B represents the result on day 14. Figure 5 C represents the result on day 28. Figure 5 D represents the results on day 35. The results showed that YG-2M1, YG-23M1, and YG-68M1 could significantly reduce the expression level of serum hLp(a) in mice in a dose-dependent manner.

[0210] Example 8. In vivo activity screening of siRNA conjugates 8.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered 3 mg / kg of the YG-90M1 conjugate, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before and five days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0211] 8.2 Experimental Results The in vivo activity screening results of the YG-90M1 conjugate are shown in [the table below]. Figure 6 The results showed that YG-90M1 could significantly reduce the expression level of serum hLp(a) in mice.

[0212] Example 9. In vivo activity screening of siRNA conjugates 9.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered the conjugate at a dose of 3 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and at 7, 14, 28, 42, and 56 days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0213] 9.2 Experimental Results The in vivo activity screening results of different siRNA conjugates are shown in the figure. Figure 7 The results showed that YG-2M1, YG-12M1, YG-19M1, YG-75M1, and YG-89M1 could significantly and continuously reduce the expression level of serum hLp(a) in mice.

[0214] Example 10. In vivo activity screening of siRNA conjugates 10.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered 1 mg / kg of the YG-89M6 conjugate, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and at 7, 14, and 56 days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0215] 10.2 Experimental Results The in vivo activity screening results of the YG-89M6 conjugate are shown in [the table below]. Figure 8 The results showed that YG-89M6 could significantly reduce the expression level of serum hLp(a) in mice.

[0216] Example 11. In vivo activity screening of siRNA conjugates 11.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered the conjugate at a dose of 1 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and on days 7 and 21 after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0217] 11.2 Experimental Results The in vivo activity screening results of different siRNA conjugates are shown in the figure. Figure 9 The results showed that YG-9M5, YG-12M5, and YG-89M5 could all significantly reduce the expression level of serum hLp(a) in mice.

[0218] Example 12. In vivo activity screening of siRNA conjugates 12.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered the conjugate at a dose of 1 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and seven days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0219] 12.2 Experimental Results The in vivo activity screening results of different siRNA conjugates are shown in the figure. Figure 10 The results showed that YG-2M1, YG-19M1, YG-19M3, YG-19M4, YG-19M6, YG-75M1, YG-89M1, YG-89M3, YG-89M4, and YG-89M6 could all reduce the expression level of serum hLp(a) in mice.

[0220] Example 13. In vivo activity screening of siRNA conjugates 13.1 Experimental Procedure Six- to eight-week-old female LPA transgenic mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Each mouse was subcutaneously administered the conjugate at a dose of 3 mg / kg, with three mice in each group. Blood was collected from the orbital venous plexus of the mice one day before administration and on days 3 and 7 after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit (Mercodia, catalog number: 10-1106-01).

[0221] 13.2 Experimental Results The in vivo activity screening results of different siRNA conjugates are shown in the figure. Figure 11 The results showed that YG-9M1, YG-17M1, YG-19M1, and YG-75M1 could all reduce the expression level of serum hLp(a) in mice.

[0222] Example 14. Results of the in vivo activity of siRNA conjugates in cynomolgus monkeys. 14.1 Experimental Procedure Eight male cynomolgus macaques were selected and the experiment was conducted by Junke Zhengyuan (Guangxi) Biomedical Technology Co., Ltd. After acclimatization, they were randomly divided into four groups of two macaques each according to their body weight. Each group received a single subcutaneous administration of different siRNA conjugates at a dose of 2 mg / kg. The control group received the same volume of Saline. Peripheral venous blood was collected 1 day before administration and at 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 91, 105, and 119 days after administration. Serum was separated, and the expression level of the target protein hLp(a) in the serum was detected using an ELISA kit.

[0223] 14.2 Experimental Results The in vivo activity screening results of different siRNA conjugates are shown in the figure. Figure 12 .

[0224] The siRNA conjugates YG-12M1, YG-89M1, and YG-89M6 of the present invention exhibit good activity in cynomolgus monkeys and can significantly and continuously reduce the serum hLp(a) expression level in cynomolgus monkeys.

Claims

1. A siRNA for inhibiting LPA gene expression, the siRNA comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, and the antisense strand comprising a nucleotide sequence II; each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified or unmodified nucleotide; nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region; nucleotide sequence I is substantially identical to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of at least 15 nucleotides in length in the mRNA expressing the LPA gene, preferably, the first nucleotide sequence being a nucleotide sequence of 15-25 nucleotides in length in the mRNA expressing the LPA gene, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides; Preferably, the mRNA expressing the LPA gene is as shown in NCBI refseqID NM_005577.4; specifically, the mRNA sequence is as shown in SEQ ID NO:

1.

2. The siRNA for inhibiting LPA gene expression as described in claim 1, wherein the nucleotide sequence II is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence; Preferably, in the siRNA, the sense strand and the antisense strand are of the same or different lengths, the sense strand is 16-23 nucleotides long, and the antisense strand is 19-26 nucleotides long; Preferably, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 23, or 19 / 24. Preferably, in the siRNA, the nucleotide sequence I has at least 70%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity with the first nucleotide segment; Preferably, in the siRNA, the nucleotide sequence I comprises at least 15 consecutive nucleotides differing by 0, 1, 2 or 3 nucleotides from SEQ ID NO: 2-116, such as at least 15, 16, 17, 18, 19, 20 or 21 nucleotides; preferably, the nucleotide sequence I is as shown in SEQ ID NO: 2-116; Preferably, in the siRNA, the nucleotide sequence II comprises at least 15 consecutive nucleotides differing from those in SEQ ID NO: 117-231 by 0, 1, 2 or 3 nucleotides, such as at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides; preferably, the nucleotide sequence II is as shown in SEQ ID NO: 117-231; More preferably, in the siRNA, the nucleotide sequence I is as shown in SEQ ID NO: 3, 10, 13, 18, 20, 24, 69, 76, 90 and 99, respectively, and the nucleotide sequence II is as shown in SEQ ID NO: 118, 125, 128, 133, 135, 139, 184, 191, 205 and 214, respectively.

3. The siRNA for inhibiting LPA gene expression as described in claim 1 or 2, wherein each nucleotide in nucleotide sequence I and nucleotide sequence II is a modified nucleotide, and the modified nucleotide is a fluorinated modified nucleotide or a non-fluorinated modified nucleotide; Preferably, the fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2'-position of the ribosome with fluorine, and the non-fluorinated nucleotide refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2'-position of the ribosome with a non-fluorinated group; preferably, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2'-position of the ribosome with a non-fluorinated group, and these nucleotides or nucleotide analogs may be selected from one of 2'-alkoxy-modified nucleotides or nucleotide analogs, 2'-substituted alkoxy-modified nucleotides or nucleotide analogs, 2'-alkyl-modified nucleotides or nucleotide analogs, 2'-substituted alkyl-modified nucleotides or nucleotide analogs, 2'-amino-modified nucleotides or nucleotide analogs, 2'-substituted amino-modified nucleotides or nucleotide analogs, and 2'-deoxynucleotides; Preferably, one or more nucleotides at positions 5, 7, 8, 9, 10, 11, and 12 of nucleotide sequence I are fluorinated nucleotides, in the direction from the 5' end to the 3' end; and one or more nucleotides at positions 2, 3, 5, 6, 7, 8, 9, 10, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, in the direction from the 5' end to the 3' end. Preferably, the nucleotides at positions 7, 9, 10, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 8, 9, 12, 14, and 16 of nucleotide sequence II are fluorinated nucleotides in the direction from the 5' end to the 3' end. Preferably, the nucleotides at positions 7, 9, 10, 11, and 12 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, 8, 9, and 10 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 3, 5, 7, 10, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 7, 9, and 11 of nucleotide sequence I are fluorinated nucleotides, arranged from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides, arranged from the 5' end to the 3' end; or Preferably, the nucleotides at positions 5, 7, and 9 of nucleotide sequence I are fluorinated nucleotides in the direction from the 5' end to the 3' end; and the nucleotides at positions 2, 6, 9, 14, and 16 of nucleotide sequence II are fluorinated nucleotides in the direction from the 5' end to the 3' end. Preferably, in the direction from the 5' end to the 3' end, the nucleotide sequence II contains at least one (e.g., one, two, three, four, five or more) heat-stabilizing modification at positions 4-8; preferably, it contains one heat-stabilizing modification at positions 6, 7 or 8; preferably, the heat-stabilizing modification may include, but is not limited to, debasement modification, or mismatch with a relative nucleotide in the opposite chain, or sugar modification such as 2' Deoxygenated or acyclic nucleotides, or those with impaired WC H pairs that are complementary to the target mRNA. The bonded nucleotides may be modified with phosphate esters.

4. The siRNA for inhibiting LPA gene expression as described in any one of claims 1-3, wherein at least a portion of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA are phosphate ester groups with modifying groups; preferably, the phosphate ester group with modifying groups is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom. Preferably, the thiophosphate group linkage is present at least one of the following positions: between the first and second nucleotides of the sense and / or antisense strands; between the second and third nucleotides of the sense and / or antisense strands; between the 19th and 20th nucleotides of the antisense strand; between the 20th and 21st nucleotides of the antisense strand; between the 21st and 22nd nucleotides of the antisense strand; between the 22nd and 23rd nucleotides of the antisense strand; or any combination thereof.

5. The siRNA for inhibiting LPA gene expression as described in any one of claims 1-4, wherein the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a nucleotide modified with a 5'-phosphate analog; preferably, it is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

6. An siRNA conjugate comprising any of the siRNAs described above and a conjugate group conjugated to the siRNA; preferably, the pharmaceutically acceptable conjugate group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein, The galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent; preferably, the conjugation site of siRNA and the conjugate group can be at the 3' or 5' end of the sense strand of siRNA, at the 3' end of the antisense strand, or in the internal sequence of siRNA. Preferably, in the above siRNA conjugate, the conjugating group is L96, with the structure shown below: Formula (I).

7. The siRNA conjugate as described in claim 6, The positive strand of the siRNA conjugate is selected from the nucleotide sequence shown in the following formula: 5'-XmsXmsXmXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXfXmXmXfXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXmXmXm XmXm-L96-3' or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3' or 5'-XmsXmsXmXmXmXfXmX fXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3', or 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3' The antisense strand in the siRNA conjugate is selected from the nucleotide sequence shown in the following formula: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmfXmXfXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmfXmXfXmXmXmXmXmXmsXms-3', or 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXm-3', or 5'-VPXmsXfsXmXmXmXmXf ... mXfXmXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXmXmXf sXmXmXmXfXmXfXmXmXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXm XmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXmXfXmXfXfXmXmXfXmXfXmXfXmXmXm XmXfXmXfXfXmXmXfXmXfXmXfXmXmXmXmsXmsXm-3', or 5'-VPXmsXfsXmXmXm XmsXm - 3', or 5' - VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXfXmXfXmXmXmsXmsXm - 3', or 5' - VPXms mXfXmXmXmXfXmXfXmXmXmsXmsXm-3', or 5'-VPXmsXfsXfXmXfXmXfXmXmXfXmXmXmXfXmXfXmXmXmXmXmsXmsXm-3'; in, Xm represents any nucleotide modified with 2'-methoxy, such as 2'-methoxy-modified C, G, U, A, T; Xf represents any nucleotide modified with 2'-fluoride, such as 2'-fluoride-modified C, G, U, A, T; lowercase s indicates that the two nucleotides adjacent to the letter s on the left and right are linked by thiophosphate subunits; VP indicates that the nucleotide adjacent to the right of the letter combination VP is a nucleotide modified with vinylphosphonate (5'-(E)-vinylphosphonate, E-VP).

8. The siRNA conjugate of claim 7, wherein the sense and antisense strands in the conjugate are a combination of the following: M-1： Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmXmXmsXmsXm-3'; Or M-1': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXmXmXmXfXmXmXmXmXmsXmsXm-3'; Or M-2: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmXmXmsXmsXm-3'; Or M-2': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXmXmXmXmXmXmXmXmXmXmXfXmXfXmXmXmXmsXmsXm-3'; Or M-3: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXfXmXmXmXmXmXmXm-3'; or M-3' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXmXmXmXmXmXmXfXmXmXmXmXm-3'; Or M-4: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXmXmXfXmXmXfXmXmXmXmXmXmXmsXmsXm-3'; or M-4' Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXfXmXfXmXmXfXmXmXfXmXmXmXmXmsXmsXm-3'; Or M-5: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3'; Or M-5': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXfXfXfXfXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXfXmXfXmXmXmXfXmXmXmXmXmXmXmXmXmXmXmXmXmsXmsXm-3'; Or M-6: Chain of Justice: 5'-XmsXmsXmXmXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXmXmXmXmsXmsXm-3'; Or M-6': Chain of Justice: 5'-XmsXmsXmXmXfXmXfXmXfXmXmXmXmXmXmXmXmXmXm-L96-3'; Antonym chain: 5'-VPXmsXfsXmXmXmXfXmXmXfXmXmXmXmXmXfXmXmXmXmXm-3'; Preferably, the siRNA conjugate comprises a sense strand and an antisense strand, wherein the sense strand comprises the sense strand of any of the siRNA conjugates shown in Table 2 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate; More preferably, the siRNA conjugate comprises a sense strand and an antisense strand, wherein the sense strand comprises the sense strands of YG-2M1, YG-9M1, YG-12M1, YG-17M1, YG-19M1, YG-23M1, YG-68M1, YG-75M1, YG-89M1, YG-98M1, YG-2M2, YG-12M2, YG-23M2, YG-89M2, YG-98M2, YG-89M3, YG-89M4, YG-9M5, YG-12M5, YG-89M5, and YG-89M6, the nucleic acid sequences of which are shown in SEQ ID. As shown in SEQ ID NOs 234, 235, 237, 238, 240, 242, 251, 252, 256, 259, 262, 263, 264, 265, 266, 268, 270, 271, 272, 273, and 275, the antisense strand comprises the antisense strand of the corresponding siRNA conjugate, and its nucleic acid sequences are shown in SEQ ID NOs 277, 278, 280, 281, 283, 285, 294, 295, 299, 302, 305, 306, 307, 308, 309, 311, 313, 314, 315, 316, and 318, respectively.

9. A composition comprising the siRNA according to any one of claims 1-5 or the siRNA conjugate according to any one of claims 6-8; Preferably, the composition is a pharmaceutical composition and further includes a pharmaceutically acceptable carrier or excipient.

10. Use of the siRNA of any one of claims 1-5, or the siRNA conjugate of any one of claims 6-8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and / or treatment of a disease associated with elevated Lp(a) particle levels; Preferably, the diseases associated with elevated Lp(a) particle levels include ASCVD; The diseases involved in ASCVD include, but are not limited to: coronary heart disease, cerebrovascular disease, familial hypercholesterolemia, kidney disease, diabetes, hypothyroidism, and peripheral vascular disease. Coronary heart disease includes chronic coronary artery disease, also known as chronic myocardial ischemia syndrome, which includes stable angina, ischemic cardiomyopathy, and occult coronary heart disease. Acute coronary syndrome includes unstable angina, non-ST-segment elevation myocardial infarction, and ST-segment elevation myocardial infarction. Cerebrovascular diseases are broadly classified into two categories: ischemic cerebrovascular diseases and hemorrhagic cerebrovascular diseases. Ischemic cerebrovascular diseases include, but are not limited to, cerebral infarction and transient ischemic attacks. Hemorrhagic cerebrovascular diseases include, but are not limited to, cerebral hemorrhage and subarachnoid hemorrhage. Kidney diseases include chronic kidney disease (CKD) or nephrotic syndrome. Peripheral vascular diseases include, but are not limited to, lower extremity arteriosclerosis obliterans, vasculitis, arterial embolism, arterial thrombosis, venous thrombosis, carotid and vertebral artery stenosis, pulmonary embolism, renal artery stenosis, aortic dissection aneurysm, thoracic and abdominal aortic aneurysm, Budd-Chiari syndrome, etc.

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