Double-stranded oligonucleotides that inhibit LPA gene expression and their applications

Abstract

The present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression and its application, belonging to the field of nucleic acid drug technology. The conjugate containing the double-stranded oligonucleotide provided by the present disclosure can significantly reduce the LPA protein level in the serum of cynomolgus monkeys, and the drug effect is long-lasting, and can effectively alleviate, prevent and / or treat diseases or conditions mediated by the mRNA level of LPA gene expression, and has good application prospects.

Description

Technical Field

[0001] The present disclosure relates to the technical field of nucleic acid drugs, and in particular to a double-stranded oligonucleotide for inhibiting LPA gene expression and an application thereof. Background Art

[0002] Lipoprotein (a) (Lp(a), LPA) is a low-density lipoprotein variant. It is composed of a high molecular weight glycoprotein Apo(a) subtype homologous to plasminogen (PLG) and LDL-like particles covalently bound by disulfide bonds. It is a low-density lipoprotein-like protein with an apo(a) group. LPA is the name of the gene encoding apolipoprotein (a) (apo(a)), which is mainly expressed in the liver, and its expression is limited to humans and non-primates. Plasma Lp(a) concentration is largely determined by the LPA gene, which plays a key role in revealing the causal relationship between Lp(a) and cardiovascular disease. Lipoprotein (a) levels are the direct pathogenic factor of many cardiovascular diseases and are closely related to cardiovascular diseases such as myocardial infarction, lower limb arterial disease, peripheral arteriosclerosis, and aortic valve stenosis. Mendelian randomization studies have confirmed that elevated Lp(a) levels are one of the causes of cardiovascular disease. Lp(a) is continuously associated with the risk of cardiovascular disease. Among 2,100 candidate genes for cardiovascular disease, it was found that the variation of the LPA gene is the strongest cardiovascular genetic risk factor. Therefore, the etiological role of Lp(a) in cardiovascular disease has become a research hotspot.

[0003] Based on the RNA interference mechanism, small interfering RNA (siRNA) can inhibit or block the expression of target genes in a sequence-specific manner, thereby achieving the purpose of treating diseases. Therefore, by inhibiting the expression of the LPA gene, diseases caused by abnormal lipoprotein (a) levels, especially cardiovascular diseases, can be prevented and treated at the cellular level. The guidelines for the prevention and treatment of dyslipidemia in adults define >30 mg / d1 as abnormal Lp(a). Based on this standard, about 30% of patients with previous cardiovascular events have abnormal Lp(a). Although elevated Lp(a) levels are common, there is a lack of targeted therapeutic drugs. Therefore, the development of siRNA drugs that inhibit LPA gene expression and can treat diseases related to LPA gene expression will help reduce adverse cardiovascular events.

[0004] In view of this, the present disclosure is proposed. Summary of the invention

[0005] In response to the needs of cardiovascular and cerebrovascular diseases, the present disclosure uses LPA gene as a target to develop a small nucleic acid molecule drug that can reduce cholesterol levels and thus prevent or control metabolic syndrome. The first purpose of the present disclosure is to provide a double-stranded oligonucleotide and its conjugate, pharmaceutical composition, kit for inhibiting LPA gene expression, and methods and uses thereof for inhibiting or reducing LPA gene expression or treating LPA gene-mediated diseases or symptoms.

[0006] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0007] In the first aspect of the present disclosure, the present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression, the double-stranded oligonucleotide comprising a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence I, the antisense strand comprising a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II respectively consisting of 17-21 nucleotides; wherein the nucleotide sequence I and the nucleotide sequence II are equal in length and are substantially complementary to form a duplex region, the substantial complementarity means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides, the nucleotide sequence I is a nucleotide sequence consisting of 17-19 consecutive nucleotides in the mRNA expressed by the LPA gene; characterized in that the antisense strand further comprises a nucleotide sequence III consisting of 1-3 nucleotides.

[0008] At least one of the nucleotide sequence III is a double-substituted modified nucleotide, wherein the double-substituted modified nucleotide refers to a nucleotide in which the hydroxyl group and hydrogen of the ribose 2' position of the nucleotide are replaced by [2'-R1-2'-R2]. Compared with an siRNA in which the nucleotides at the corresponding positions are unmodified nucleotides, the stability of the siRNA containing the double-substituted modified nucleotide is increased.

[0009] In some specific embodiments of the present disclosure, the nucleotide sequence III is two nucleotides in length and is located at positions 20-21 of the antisense strand in the direction from the 5' end to the 3' end;

[0010] In some specific embodiments of the present disclosure, R1 and R2 are each independently selected from halogen, substituted / unsubstituted C1-C6 alkyl or substituted / unsubstituted C1-C6 alkoxy.

[0011] In some specific embodiments of the present disclosure, the disubstituted modified nucleotide is a nucleotide in which the hydroxyl group and hydrogen at the 2'-position of ribose are substituted with [2'-F-2'-methyl].

[0012] In some specific embodiments of the present disclosure, the antisense strand comprises a nucleotide sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21 consecutive nucleotides of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16 in Table 1, or comprises a nucleotide sequence with 1, 2 or 3 nucleotide differences between the consecutive nucleotides; and / or, the sense strand comprises a nucleotide sequence of at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleotides of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15 in Table 1, or comprises a nucleotide sequence with 1, 2 or 3 nucleotide differences between the consecutive nucleotides.

[0013] In some specific embodiments of the present disclosure, the antisense strand comprises any one of the sequences shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16, or a nucleotide sequence having 1 or 2 nucleotide differences from any of the above sequences.

[0014] In some specific embodiments of the present disclosure, the sense strand comprises any one of the sequences shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13 and 15, or a nucleotide sequence having 1 or 2 nucleotide differences from any of the above sequences.

[0015] In some specific embodiments of the present disclosure, the antisense strand comprises any one of the sequences shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14 or 16, and the sense strand comprises any one of the sequences shown in SEQ ID NO. 1, 3, 5, 7, 9, 11, 13 or 15.

[0016] In some specific embodiments of the present disclosure, the double-stranded oligonucleotide is selected from one or more of the following groups:

[0017] 1) The antisense strand has the nucleotide sequence shown in SEQ ID NO.2, and the sense strand has the nucleotide sequence shown in SEQ ID NO.1;

[0018] 2) the antisense strand has the nucleotide sequence shown in SEQ ID NO.4, and the sense strand has the nucleotide sequence shown in SEQ ID NO.3;

[0019] 3) the antisense strand has the nucleotide sequence shown in SEQ ID NO.6, and the sense strand has the nucleotide sequence shown in SEQ ID NO.5;

[0020] 4) the antisense strand has the nucleotide sequence shown in SEQ ID NO.8, and the sense strand has the nucleotide sequence shown in SEQ ID NO.7;

[0021] 5) the antisense strand has the nucleotide sequence shown in SEQ ID NO.10, and the sense strand has the nucleotide sequence shown in SEQ ID NO.9;

[0022] 6) the antisense strand has the nucleotide sequence shown in SEQ ID NO.12, and the sense strand has the nucleotide sequence shown in SEQ ID NO.11;

[0023] 7) The antisense strand has the nucleotide sequence shown in SEQ ID NO.14, and the sense strand has the nucleotide sequence shown in SEQ ID NO.13;

[0024] 8) The antisense strand has the nucleotide sequence shown in SEQ ID NO.16, and the sense strand has the nucleotide sequence shown in SEQ ID NO.15.

[0025] Table 1

[0026]

[0027] In some embodiments of the present disclosure, the double-stranded oligonucleotide may further contain modified nucleotides as required, and the modified nucleotides will not significantly weaken or lose the function of the siRNA in inhibiting the expression of the LPA gene.

[0028] In some embodiments of the present disclosure, at least one nucleotide in the sense strand or the antisense strand of the double-stranded oligonucleotide is a modified nucleotide, for example, the modified nucleotide is a nucleotide group in which the ribose group and optionally the phosphate group are modified, but is not limited thereto.

[0029] In an optional embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides. Wherein, "all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides" means that most but not all of the nucleotides in the double-stranded oligonucleotide are modified nucleotides, and may contain no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0030] In an optional embodiment of the present disclosure, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and the modification includes one or both of a single substitution or a double substitution modification at the 2' position of the ribose group of the nucleotide.

[0031] Among them, the structural formula of nucleotide is: , Base represents a nucleoside base, and the nucleoside base on each nucleotide is independently selected from uracil U, thymine T, cytosine C, adenine A or guanine G.

[0032] In an optional embodiment of the present disclosure, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides:

[0033] 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-(CH2) x -OR m Modified nucleotides, 2'-O-Si(R n )3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, quasi-nucleotides or [2'-F-2'-methyl] disubstituted modified nucleotides; the quasi-nucleotides are selected from one or more of peptide nucleic acids (PNA), morpholino nucleic acids (MNA), bridged nucleic acids (BNA), locked nucleic acids (LNA), glycol nucleic acids / glycerol nucleic acids (GNA), threose nucleic acids (TNA) or unlocked nucleic acids (UNA).

[0034] Wherein, x is selected from 1 or 2, R m Selected from substituted / unsubstituted C 1-6 Alkyl or substituted / unsubstituted C 1-6 Alkoxy, if R m Contains a substituent selected from halogen, C 1-6 Alkoxy, hydroxy or amino.

[0035] R n Independently selected from substituted / unsubstituted C 1-6 Alkyl, if R n Contains a substituent, and the substituent is selected from halogen, C1-C3 alkyl or C1-C3 alkoxy.

[0036] In the present disclosure, 2'-O-(CH2) x -R m Modified nucleotides are nucleotides in which the hydrogen atom on the 2' hydroxyl group of the ribose group is replaced by -(CH2) x -R m Wherein, when x is selected from 1, the 2'-O-(CH2) x -Rm The modified nucleotide is selected from a 2'-O-ethoxymethyl modified nucleotide or a 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide. When x is selected from 2, the 2'-O-(CH2) x -R m The modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides (also known as 2'-O-moe modified nucleotides).

[0037] In some specific embodiments of the present disclosure, the 2'-O-(CH2) x -OR m The modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides or 2'-O-ethoxymethyl modified nucleotides;

[0038] In the present disclosure, “2′-O-Si(R n )3 modified nucleotide refers to a nucleotide in which the hydrogen atom on the 2' hydroxyl group of the ribose group is replaced by -Si(R n )3 replaces the formed nucleotide. Exemplarily, a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide; wherein the structural formula of TBDMS is The structural formula of TIPS is , the structural formula of TOM is .

[0039] In some specific embodiments of the present disclosure, the 2'-O-Si(R n )3 modified nucleotides are selected from 2'-O-TBDMS modified nucleotides, 2'-O-TIPS modified nucleotides or 2'-O-TOM modified nucleotides.

[0040] In some specific embodiments of the present disclosure, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, wherein the modification includes a single or double substitution modification at the 2' position of the ribose group of the nucleotide, and the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: 2'-fluorine-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, [2'-F-2'-methyl] double-substituted modified nucleotides, or a combination of two thereof.

[0041] In some specific embodiments of the present disclosure, the double-stranded oligonucleotide contains at least one 2'-O-methoxyethyl modified nucleotide.

[0042] In some specific embodiments of the present disclosure, the antisense strand contains at least one 2'-O-methoxyethyl modified nucleotide.

[0043] In some specific embodiments of the present disclosure, from the 5' end to the 3' end, the 20th-21st nucleotides of the antisense strand are disubstituted modified nucleotides, and the disubstituted modified nucleotides are selected from nucleotides in which the 2' hydroxyl group and hydrogen of the ribose are disubstituted by [2'-F-2'-methyl].

[0044] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and / or, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, any one of the nucleotides at positions 9, 10, 11 and 12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotides at positions 20-21 are selected from nucleotides in which the 2'-hydroxyl and hydrogen of the ribose are disubstituted with [2'-F-2'-methyl], and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0045] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 9, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, the nucleotides at positions 20-21 are selected from nucleotides in which the 2'-hydroxyl and hydrogen of the ribose are disubstituted with [2'-F-2'-methyl], and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0046] In some specific embodiments of the present disclosure, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate internucleotide linkages.

[0047] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least one of the following linkages between nucleotides of the sense strand is a phosphorothioate linkage:

[0048] (1) the connection between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

[0049] (2) The connection between the second nucleotide and the third nucleotide at the 5' end of the sense strand.

[0050] In some specific embodiments of the present disclosure, in the direction from the 5' end to the 3' end, at least one of the following linkages between nucleotides of the antisense strand is a phosphorothioate linkage:

[0051] (1) the connection between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

[0052] (2) a connection between the second nucleotide and the third nucleotide at the 5' end of the antisense strand;

[0053] (3) a connection between the first nucleotide and the second nucleotide at the 3' end of the antisense strand;

[0054] (4) The connection between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.

[0055] In some optional embodiments of the present disclosure, the sense strand or the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0056] In some optional embodiments of the present disclosure, the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0057] In some alternative embodiments of the present disclosure, the antisense strand comprises a 3' overhang of 2 nucleotides.

[0058] In some specific embodiments of the present disclosure, the sense strand comprises or is selected from any one of the modified nucleotide sequences shown in A1) to A8).

[0059] Specifically, from the 5' end to the 3' end, the modified nucleotide sequences shown in A1)-A8) are as follows:

[0060] A1: CmsUmsGmGmCmUmUfGfAfUfCmAmAmGmAmAmCmUmAm;

[0061] A2: CmsCmsUmAmGmAmGfGfCfUfCmCmUmUmCmCmGmAmAm;

[0062] A3: CmsAmsGmGmAmCmUfGfCfUfAmCmUmAmCmCmAmUmUm;

[0063] A4: GmsAmsAmUmCmCmAfGfAfUfGmCmUmGmAmGmAmUmUm;

[0064] A5: GmsGmsAmAmGmGmAfCfAfUfGmUmCmAmGmUmCmUmUm;

[0065] A6: AmsGmsAmGmGmAmCfAfAfCfAmGmAmAmUmAmUmUmAm;

[0066] A7: GmsGmsAmAmGmAmAfCfAfUfGmUmCmAmGmUmCmUmUm;

[0067] A8: GmsGmsAmAmGmGmAfCfAfUfGmUmCmAmAmUmCmUmUm.

[0068] In some specific embodiments of the present disclosure, the antisense strand comprises or is selected from any one of the modified nucleotide sequences shown in B1) to B8).

[0069] Specifically, from the 5' end to the 3' end, the modified nucleotide sequences shown in B1)-B8) are as follows:

[0070] B1: UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfGmCfCmAmGms(NM054)s(NM054);

[0071] B2: UmsUfsCmGmGmAfAmGmGfAmGmCmCmUfC(moe)UfAmGmGms(NM054)s(NM054);

[0072] B3: AmsAfsUmGmGmUfAmGmUfAmGmCmAmGfU(moe)CfCmUmGms(NM054)s(NM054);

[0073] B4: AmsAfsUmCmUmCfAmGmCfAmUmCmUmGfG(moe)AfUmUmCms(NM054)s(NM054);

[0074] B5: AmsAfsGmAmCmUfGmAmCfAmUmGmUmCfC(moe)UfUmCmCms(NM054)s(NM054);

[0075] B6: UmsAfsAmUmAmUfUmCmUfGmUmUmGmUfC(moe)CfUmCmUms(NM054)s(NM054);

[0076] B7: AmsAfsGmAmCmUfGmAmCfAmUmGmUmUfC(moe)UfUmCmCms(NM054)s(NM054);

[0077] B8: AmsAfsGmAmUmUfGmAmCfAmUmGmUmCfC(moe)UfUmCmCms(NM054)s(NM054).

[0078] In some specific embodiments of the present disclosure, the double-stranded oligonucleotide comprises one or more of the groups RX301078-RX301085 in Table 2:

[0079] Table 2

[0080]

[0081] Wherein, C, G, U, A, T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, thymidine-3'-phosphate, respectively; m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl modified nucleotide; f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorine modified nucleotide; (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl modified nucleotide; s represents that the two nucleotides are connected by a thiophosphate backbone;

[0082] (NM054) represents a nucleotide in which the hydroxyl group and hydrogen at the 2' position of the ribose are replaced by [2'-F-2'-methyl], and the structural formula is .

[0083] In an optional embodiment, the double-oligonucleotide is selected from siRNA.

[0084] In a second aspect of the present disclosure, the present disclosure provides a conjugate, which comprises the double-stranded oligonucleotide according to the first aspect of the present disclosure, and one or more ligands capable of binding to a cell surface receptor conjugated to the double-stranded oligonucleotide.

[0085] The conjugate has a structure represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0086]

[0087] Formula (I)

[0088] In formula (I), m is selected from 1, 2, 3 or 4;

[0089] In some specific embodiments of the present disclosure, each Z is hydroxyl.

[0090] In some embodiments of the present disclosure, each p is independently 1 or 2.

[0091] In some specific embodiments of the present disclosure, each p is 1.

[0092] In some embodiments of the present disclosure, each q is independently 1 or 2.

[0093] In some specific embodiments of the present disclosure, each q is 1.

[0094] In some specific embodiments of the present disclosure, each p is 1, and each q is 1.

[0095] In some specific embodiments of the present disclosure, each R3 is H.

[0096] In some specific embodiments of the present disclosure, each Y is O.

[0097] Each L is independently selected from C1-C 30 Alkylene or ; where each R L2a Each independently selected from C1-C 10 Alkylene, each R L2b are each independently selected from O, S, NH or -NH-C(O)-, and k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0098] Further, in some specific embodiments of the present disclosure, the conjugate has a structure as shown in formula (II) or a pharmaceutically acceptable salt thereof:

[0099]

[0100] Formula (II)

[0101] In formula (II), m is selected from 1, 2 or 3; L is independently selected from -CH2-, -CH2-CH2-, -CH2-CH2-CH2-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -, -CH2-NH-CO-(CH2)5-, -(CH2)2-NH-CO-(CH2)4-, -(CH2)3-NH-CO-(CH2)3-, -(CH2)4-NH-CO-(CH2)2-, or -(CH2)5-NH-CO-CH2-.

[0102] In some alternative embodiments of the present disclosure, each L2 is independently selected from -CH2-CH2- or -(CH2)2-NH-CO-(CH2)4-.

[0103] In some specific embodiments of the present disclosure, L2 is selected from -CH2-CH2-.

[0104] In some specific embodiments of the present disclosure, L2 is selected from -(CH2)2-NH-CO-(CH2)4-.

[0105] In some specific embodiments of the present disclosure, the conjugate has a structure shown in the following formula (III) or formula (IV) or a pharmaceutically acceptable salt thereof:

[0106] or .

[0107] Nu represents the double-stranded oligonucleotide according to the first aspect of the present disclosure, and the 3' end and / or 5' end of the sense strand and / or antisense strand of the double-stranded oligonucleotide is conjugated to the ligand.

[0108] In some specific embodiments of the present disclosure, the 3' end and / or the 5' end of the sense strand is conjugated to the ligand.

[0109] In some specific embodiments of the present disclosure, the 3' end of the sense strand is conjugated to the ligand.

[0110] In some specific embodiments of the present disclosure, the conjugate includes one or more of the groups R301078-R301085 in Table 3:

[0111] Table 3

[0112]

[0113] Exemplary explanation, "_(CR01008×3)" indicates that the ligand represented by the formula (CR01008×3) is conjugated to the 3' end of the indicated sense strand.

[0114] In the third aspect of the present disclosure, the present disclosure provides a pharmaceutical composition, which comprises the double-stranded oligonucleotide described in the first aspect of the present disclosure or the conjugate described in the second aspect of the present disclosure.

[0115] In some optional embodiments of the present disclosure, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0116] The pharmaceutical compositions disclosed herein include preparations suitable for parenteral administration. The preparations can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with excipients to prepare a single dose form is generally the amount of siRNA that produces a therapeutic effect.

[0117] In the fourth aspect of the present disclosure, the present disclosure provides a use of the double-stranded oligonucleotide described in the first aspect of the present disclosure, the conjugate described in the second aspect of the present disclosure, or the pharmaceutical composition described in the third aspect of the present disclosure in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene.

[0118] In some optional embodiments of the present disclosure, the LPA gene-mediated disease or condition includes cardiovascular disease, including but not limited to hyperlipoprotein (a) blood, Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic stenosis and / or any other disease or pathology associated with elevated levels of Lp (a) particles.

[0119] In a fifth aspect of the present disclosure, the present disclosure provides a kit comprising the double-stranded oligonucleotide described in the first aspect of the present disclosure, the conjugate described in the second aspect of the present disclosure, or the pharmaceutical composition described in the third aspect of the present disclosure.

[0120] Compared with the prior art, the present invention has the following beneficial effects:

[0121] The double-stranded oligonucleotides, conjugates and pharmaceutical compositions provided by the present disclosure can significantly reduce the LPA protein level in the serum of cynomolgus monkeys, and the drug effect is long-lasting. This shows that the double-stranded oligonucleotides, conjugates and pharmaceutical compositions provided by the present disclosure can effectively regulate the LPA level in serum, and can effectively alleviate, prevent and / or treat diseases or conditions mediated by the mRNA level expressed by the LPA gene, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 The change level of LPA protein in the serum of cynomolgus monkeys after administration of siRNA conjugates in Example 1. DETAILED DESCRIPTION

[0123] The technical solution of the present disclosure will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

[0124] Explanation of terms

[0125] In the present disclosure, the terms "apolipoprotein (a) gene", Apo (a) gene, and LPA are used interchangeably in the present disclosure. LPA includes but is not limited to human LPA, cynomolgus monkey LPA, mouse LPA, and rat LPA, and their amino acids, complete coding sequences, and mRNA sequences are easily obtained using publicly available databases; their amino acids, complete coding sequences, and mRNA sequences may be naturally occurring, synthetic, recombinant, or any combination thereof.

[0126] In the present disclosure, the terms “comprise” or “include” are open expressions, that is, including the contents specified in the present disclosure but not excluding other contents.

[0127] In this disclosure, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0128] In the present disclosure, the hydroxyl group and hydrogen at the 2'-position of the ribose of the nucleotide are substituted by [2'-R1-2'-R2], which means that the 2'-position of the ribose of the nucleotide is substituted by both the R1 substituent and the R2 substituent.

[0129] In the present disclosure, the term "small interfering RNA (siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates targeted cleavage of RNA transcripts in the RISC pathway by forming a silencing complex (RISC). Specifically, siRNA guides the specific degradation of mRNA sequences through the known RNA interference (RNAi) process, inhibiting the translation of mRNA into amino acids and proteins. For example, siRNA can regulate (e.g., inhibit) the expression of LPA in cells.

[0130] In the present disclosure, the term "complementary" refers to the ability of an oligonucleotide of a first sequence to hybridize with an oligonucleotide of a second sequence under certain conditions and form a double-stranded structure.

[0131] In the present disclosure, the term "nucleotide difference" and the term "nucleotide base difference" and the term "nucleotide sequence difference" can be used interchangeably. It means that compared with the original nucleotide sequence, the base type of the nucleotide at the same or corresponding position has changed. For example, when one nucleotide base in the original nucleotide sequence is A, when the nucleotide base at the same or corresponding position is changed to U, C, G or dT, dC, dG, etc., it is considered that there is a difference in the nucleotide sequence at this position. It should be noted here that, when compared with the original nucleotide sequence, the nucleotide at the same or corresponding position only differs in the presence or absence of modification or the type of modification, it is not considered that there is a difference in the nucleotide sequence at this position.

[0132] In the present disclosure, the term "overhang" refers to at least one unpaired nucleotide protruding from the double-stranded oligonucleotide double helix structure, and is also a nucleotide sequence in the siRNA structure other than the double-stranded region. For example: when the 3' end of one strand in the sense strand and / or the antisense strand exceeds the 5' end of the other strand, or when the 5' end of one strand in the sense strand and / or the antisense strand exceeds the 3' end of the other strand, there is a nucleotide overhang. The overhang may contain at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more nucleotides. The nucleotide overhang may contain or be composed of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be located in the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may appear at the 5' end, the 3' end, or both ends of the antisense or sense strand.

[0133] In the present disclosure, the term "inhibiting the expression of the LPA gene" includes any level of inhibition of the LPA gene, for example, at least partial inhibition of LPA gene expression, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. Wherein, the expression of the LPA gene can be evaluated based on the level of any variable related to the expression of the LPA gene, for example, the mRNA level or protein level of LPA. Inhibition can be evaluated by a decrease in the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level utilized in the art, e.g., a baseline level prior to administration, or a level measured from a similar subject, cell, or sample that has not been treated or that has been treated with a control (e.g., a buffer-only control or a no active agent control).

[0134] In the present disclosure, "conjugation" refers to the connection between two or more chemical moieties by covalent bonding; "conjugate" refers to a compound formed by covalent bonding between chemical moieties; "conjugated molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide through a reaction to ultimately form the oligonucleotide conjugate of the present disclosure.

[0135] In the present disclosure, "pharmaceutical composition" may refer to a composition used for the treatment of a disease, and may also be used for in vitro culture experiments of cells. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with an excipient that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and fully combining the active siRNA with a liquid excipient, a finely divided solid excipient, or both.

[0136] In the present disclosure, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammals treated therewith. Preferably, the "pharmaceutically acceptable" described in the present disclosure refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, especially humans.

[0137] In the present disclosure, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the siRNA of the present disclosure, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present disclosure.

[0138] In addition to any conventional excipients, to the extent that they are incompatible with the siRNA of the present disclosure, such as producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present disclosure.

[0139] The present disclosure is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present disclosure in any form.

[0140] Unless otherwise specified, the siRNA sequences used in the present disclosure were commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the PCR primers used in the present disclosure were commissioned to Sangon Biotechnology (Shanghai) Co., Ltd. for synthesis.

[0141] The detection data of Apo(a) protein at different time points in the in vivo activity experiment involved in the present disclosure and the calculation method are summarized as follows:

[0142] ;

[0143] In the context of this disclosure, unless otherwise stated, in vivo activity experimental data are presented as The experimental data were plotted and analyzed using GraphPad prism 8.0 software.

[0144] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0145] Preparation of compounds

[0146] Unless otherwise specified, the reagents used in the preparation of the compounds disclosed herein were purchased from Beijing Coupling Technology Co., Ltd. The information of the main reagents is shown in Table 4.

[0147] Table 4

[0148]

[0149] Among them, CPG stands for controlled aperture glass (Controlled Pore Glass) carrier.

[0150] Preparation Example 1 Preparation of Compound CR01008

[0151] (1.1) The synthetic structure of compound CR01008 is shown below:

[0152]

[0153] The synthetic route of compound CR01008 is as follows:

[0154] .

[0155] (1.1.1) Synthesis of compound 2

[0156] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde aqueous solution (8.9 g, 37 mass%, 2.4 eq) were dissolved in 33 ml of methanol, and 13 ml of KOH aqueous solution with a concentration of 45.3 mass% was added dropwise. After the addition was completed, the reaction was stirred at 25°C for 30 minutes, the temperature was raised to 60°C and refluxed at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness under reduced pressure to obtain a crude product as a white solid. A small amount of water was added to the crude product for pulping and filtration to obtain compound 2 as a white solid (9 g, yield 78. 9%). MS-ESI (m / z) = 260 [M + H] + .

[0157] (1.1.2) Synthesis of compound 3

[0158] Compound 2 (9 g, 1 eq) prepared in step (1.1.1) was dissolved in 70 ml of 1,4-dioxane, and a solution of hydrogen chloride in 1,4-dioxane (45 ml, 4 M) was added, and stirred at 25°C for 1 hour. After the reaction, the reaction solution was evaporated under reduced pressure to obtain compound 3 (6.8 g, yield 100%) as a white solid.

[0159] (1.1.3) Synthesis of compound 5

[0160] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetyloxy-6-(acetyloxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq) and DIEA ( N,N -Diisopropylethylamine, 3.5 g, 6.0 eq) was dissolved in 15 ml of DMF, HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25 °C for 3 hours in a N2 atmosphere. After the reaction, the reaction solution was evaporated under reduced pressure and purified by reverse phase column chromatography (22% acetonitrile aqueous solution) to obtain compound 5 (1.78 g, yield 64.4%) as a white solid. MS-ESI (m / z) = 589 [M + H] + .

[0161] (1.1.4) Synthesis of compound 6

[0162] Compound 5 (1.54 g, 1.0 eq) prepared in step (1.1.3) was dissolved in 15 ml of pyridine, the reaction system was cooled to 0°C with an ice-water bath, and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C, and the reaction was carried out at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After the reaction was completed, the reaction solution was evaporated under reduced pressure and purified by reverse phase column chromatography (60% by volume of acetonitrile in water) to obtain compound 6 (1 g, yield 42.7%) as a yellow solid. MS-ESI ( m / z ) = 891 [M + H] + .

[0163] (1.1.5) Synthesis of compound CR01008

[0164] Compound 6 (1.08 g, 1.0 eq) prepared in step (1.1.4) was dissolved in 20 ml of anhydrous dichloromethane, and DCI (115 mg, 0.8 eq) and compound 7 (bis(diisopropylamino)(2-cyanoethoxy)phosphine, 732 mg, 2.1 eq) were added respectively. The atmosphere was replaced with nitrogen three times and stirred at 25°C for 2 hours. After the reaction, 20 ml of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the mixture was extracted three times with 20 ml of dichloromethane (3×20 ml). The organic phases were combined and evaporated to dryness under reduced pressure. The organic phases were purified by reverse phase chromatography (72% by volume acetonitrile aqueous solution) and then vacuum dried for 12 hours to obtain compound CR01008 (1 g, yield 76.0%) as a white powder. MS-ESI (m / z) = 1091 [M + Na] + .

[0165] H NMR (400 MHz, DMSO- d 6) δ 1.05 (d, J = 6.7 Hz, 6H).1.14 (d, J = 6.7 Hz,6H), 1.37 – 1.17 (m, 5H), 1.60 – 1.40 (m, 6H),1.68 – 1.62 (m, 1H),1.80 (s,3H),1.80 (s, 3H),1.92 (s, 3H), 2.02 (s, 5H),2.13 (s, 3H),2.71 (t, J = 5.9 Hz,2H), 2.79 (d, J = 8.4 Hz, 1H), 2.87 (d, J= 8.4 Hz, 1H), 3.36 (s, 1H), 3.58 – 3.39 (m, 3H), 3.69 – 3.60 (m, 2H), 3.75 (s, 7H), 3.90 (dt, J = 11.2, 8.8 Hz, 1H),4.05 (s, 3H),4.51 (d, J = 8.4 Hz, 1H),4.99 (dd, J = 11.3, 3.4 Hz, 1H), 5.24 (d, J =3.4 Hz, 1H), 5.78 (s, 1H), 6.93 – 6.87 (m, 4H), 7.35 – 7.21 (m, 7H), 7.44 –7.37 (m, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H).

[0166] (1.2) Synthesis of compound CR01008Z

[0167]

[0168] The synthetic route of compound CR01008Z is as follows:

[0169]

[0170] (1.2.1) Synthesis of compound 9

[0171] Compound 6 (500 mg) prepared in step (1.1.4) was dissolved in 10 ml of dichloromethane, and compound 8 (succinic anhydride, 112 mg), DMAP (6.8 mg) and TEA (226.2 mg) were added. The nitrogen atmosphere was replaced three times, and the mixture was stirred at 25°C for 16 hours. The mixture was flash purified to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M + Na] + .

[0172] (1.2.2) Synthesis of compound CR01008Z

[0173] Compound 9 (50 mg, aminoCPG (1.25 g, 80 μmol / g, 0.1 mmol), HBTU (27 mg), and DIEA (12 mg) prepared in step (1.2.1) were added to a 20 ml sample bottle and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml acetonitrile (1×10 ml) and then vacuum dried. The dried filter cake, DMAP (3 mg), Cap1 (10 ml, 200 V), and Cap2 (1 ml, 20 V) were added to a 20 ml sample bottle and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was first washed once with 10 ml acetonitrile (1×10 ml), and then vacuum dried to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0174] Among them, Cap1 and Cap2 are capping reagents, Cap1 is a 20 volume % N- A pyridine / acetonitrile mixed solution of methylimidazole, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20 volume %.

[0175] Preparation Example 2 Synthesis of Compound NM054

[0176] In this preparation example, the synthesis route of compound NM054 is as follows:

[0177]

[0178] (2-1) Synthesis of compound NM054-2

[0179] Compound NM054-1 (3 g, 11.54 mmol, 1.0 eq, (2'R)-2'-deoxy-2'-fluoro-2'-methyluridine, CAS No. 863329-66-2) and pyridine (30 ml) were added to a 500 ml reactor, the temperature was lowered to 0°C, 4,4'-dimethoxytrityl chloride (4.29 g, 12.7 mmol, 1.1 eq) was added in batches, nitrogen was replaced 3 times, the reaction system was stirred at 25°C for 3 hours in a nitrogen atmosphere, and HPLC showed no raw material. After the reaction was completed, the reaction solution was concentrated, purified water (50 ml) and ethyl acetate (50 ml) were added for extraction, the organic phase was separated, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound NM054-2 (2.7 g, yield 41.7%). MS ESI (m / z) =563.0 [M + H] + .

[0180] (2-2) Synthesis of compound NM054

[0181] Compound NM054-2 (2.7 g, 4.8 mmol, 1.0 eq) was added to a 100 ml reaction kettle, and bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.74 g, 5.76 mmol, 1.2 eq) was added in batches, and then 4,5-dicyanoimidazole (0.45 g, 3.8 mmol, 0.8 eq, referred to as DCI, CAS No. 1122-28-7) and dichloromethane (27 ml) were added. The nitrogen atmosphere was replaced three times, and the reaction system was stirred at 25 ° C for 3 hours in a nitrogen atmosphere. After the reaction was completed, sodium bicarbonate aqueous solution (20 ml) was added to the reaction solution, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase column chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to obtain compound NM054 (3.0 g). MS ESI (m / z) = 763 [M + H] + .

[0182] Preparation Example 3 Preparation of siRNA

[0183] (3-1) Synthesis of the positive chain (SS)

[0184] By the phosphoramidite nucleic acid solid phase synthesis method, the compound of the solid phase carrier is linked to nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. In the synthesis process, the compound NM054 is regarded as a nucleoside monomer.

[0185] Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The synthesis conditions are given as follows:

[0186] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0187] The conditions of the deprotection reaction in each step are the same. The conditions of the deprotection reaction are: temperature of 25°C, reaction time of 70 seconds, deprotection reagent of dichloroacetic acid in dichloromethane solution (3 volume %), and the molar ratio of dichloroacetic acid to 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.

[0188] The conditions of each coupling reaction are the same. The coupling reaction conditions are as follows: temperature is 25°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:65, the reaction time is 600 seconds, the coupling reagent is a 0.5M 5-ethylthio-1H-tetrazole acetonitrile solution, and the thio reagent is a 0.2M acetonitrile / pyridine mixed solution of hydrogenated xanthanone (the volume ratio of acetonitrile and pyridine is 1:1).

[0189] The conditions of each capping reaction are the same. The conditions of the capping reaction are: temperature is 25°C; reaction time is 2 minutes; the capping reagent solution is a mixed solution of Cap1 and Cap2 with a molar ratio of 1:1, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, the volume ratio of pyridine to acetonitrile is 3:5, Cap2 is an acetonitrile solution of acetic anhydride with a volume ratio of 20 volume%; the molar ratio of N-methylimidazole in Cap1 capping reagent, acetic anhydride in Cap2 capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

[0190] The conditions for each step of the oxidation reaction are the same. The conditions for the oxidation reaction are: temperature 25°C; reaction time 3 seconds; oxidation reagent concentration is 0.05M iodine water, and the molar ratio of iodine to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 30:1; the oxidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9). The conditions for the sulfidation reaction are: temperature 25°C; reaction time 360 ​​seconds; thiolation reagent concentration is 0.2M hydrogenated xanthin pyridine solution, and the molar ratio of thiolation reagent to the nucleic acid sequence connected to the solid phase carrier in the coupling reaction is 4:1; the sulfidation reaction is carried out in a water / pyridine mixed solvent (the volume ratio of water to pyridine is 1:9).

[0191] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0192] The cleavage and deprotection conditions are as follows: the synthesized nucleotide sequence connected to the solid phase carrier is added to 25% by mass ammonia water, the amount of ammonia water is 0.5 ml / μmol, the reaction is carried out at 55°C for 16 hours, the solvent is removed, and the product is concentrated to dryness in vacuo. After the ammonia water treatment, the product is dissolved with 0.4 ml / μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride are added to remove the 2'-O-TBDMS protection on the ribose.

[0193] Purification and desalting conditions: The nucleic acid was purified by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, eluent 1 was 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); eluent 2 was 1.5 M sodium chloride, 20 mM sodium phosphate (pH = 8.1), and the solvent was a water / acetonitrile mixed solution (the volume ratio of water to acetonitrile was 9:1); the elution gradient was eluent 1: eluent 2 = (100:0)-(50:50). The product eluates were collected and combined, and desalted using a reverse phase chromatography purification column. The desalting conditions included desalting using a dextran gel column, the filler was dextran gel G25, and eluted with deionized water.

[0194] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the positive chain of siRNA is obtained.

[0195] (3-2) Synthesis of antisense strand (AS)

[0196] The antisense chain was synthesized using a universal solid phase carrier. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the solid phase synthesis method of the antisense chain are the same as those in step (3-1) for synthesizing the sense chain.

[0197] Detection: Use ion exchange chromatography (IEX-HPLC) for purity detection; use liquid chromatography-mass spectrometry (LC-MS) for molecular weight detection, and compare the measured value of the molecular weight with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the antisense chain of siRNA is obtained.

[0198] (3-3) Synthesis of siRNA

[0199] The sense chain synthesized in step (3-1) and the antisense chain synthesized in step (3-2) are mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and kept at room temperature for 10 minutes, so that the sense chain and the antisense chain form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA having the sense chain and antisense chain shown in Table 2.

[0200] Preparation Example 4 Synthesis of siRNA conjugates with the 3' end of the positive strand conjugated to a (CR01008×3) carrier

[0201] (4-1) Synthesis of the positive strand of the 3' end conjugated (CR01008×3) vector

[0202] By the method of phosphoramidite nucleic acid solid phase synthesis, starting with the compound CR01008Z connected to the solid phase carrier, the nucleoside monomers are connected one by one in a cycle in the 3'-5' direction according to the nucleotide sequence. In the synthesis process, the compound CR01008 is regarded as a nucleoside monomer, and each connection of a compound includes four steps of deprotection, coupling, capping, oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, purification and desalting conditions in the synthesis of the sense chain of this preparation example are the same as those in the synthesis of the sense chain in step (3-1) of preparation example 3.

[0203] In the synthesis process of the positive chain of the present invention, three clusters of CR01008 are obtained, which are recorded as (CR01008×3), and their structural formula is as follows:

[0204] .

[0205] (4-2) Synthesis of antisense strand

[0206] The antisense strand of this preparation example was synthesized according to the antisense strand synthesis method shown in step (3-2) of preparation example 3.

[0207] (4-3) According to the method shown in step (3-3) of Preparation Example 3, the siRNA conjugates shown in Table 3 with the 3' end of the sense strand conjugated to the (CR01008×3) vector were synthesized.

[0208] The structural formula of the siRNA conjugate of the 3' end of the positive chain conjugated to the vector (CR01008×3) is as follows:

[0209]

[0210] in, Represents siRNA, (CR01008×3) conjugated to the 3' end of the sense strand of siRNA via a phosphodiester bond.

[0211] Biological detection experiments

[0212] Unless otherwise stated, the reagents, consumables and instruments used in the biological detection experiments disclosed herein are all derived from commercially available products.

[0213] Example 1 Evaluation of the protein-lowering effect of CR01008 vector-conjugated siRNA in normal cynomolgus monkeys

[0214] In this example, the ELISA method was used to determine the expression of LPA protein in the serum of cynomolgus monkeys at different time points after a single administration of R301078, R301079, R301080, R301081, R301082, R301083, R301084, and R301085.

[0215] Animal grouping, drug administration and tissue sample collection:

[0216] Healthy cynomolgus monkeys aged 2.5-8 years were divided into groups according to serum LPA protein levels, with 3 males in each group. Each test group was given a predetermined dose of drug conjugate and a vehicle control group was added. The dosage of all animals was calculated according to body weight, and a single dose was given by subcutaneous injection in the back and / or hind limbs. Each drug conjugate was administered in the form of 3 mg (calculated as siRNA) / mL of 0.9% sodium chloride injection, and the administration volume was 1 mL / kg (cynomolgus monkey body weight), that is, the dosage of each drug conjugate was 3 mg (calculated as siRNA) / kg (cynomolgus monkey body weight). The vehicle control group was given 1 mL / kg (cynomolgus monkey body weight) of 0.9% sodium chloride injection without siRNA conjugate. Serum was collected from cynomolgus monkeys on the day of administration (recorded as Day 1, before administration), the 8th day (recorded as D8), the 15th day (recorded as D15), the 22nd day (recorded as D22), the 29th day (recorded as D29), the 36th day (recorded as D36), the 43rd day (recorded as D43), the 50th day (recorded as D50), the 57th day (recorded as D57), the 64th day (recorded as D64), the 78th day (recorded as D78), and the 92nd day (recorded as D92) after administration, and the LPA protein expression was determined using the Mercodia LPA ELISA kit (10-1106-01). Among them, the detection of the R301080, R301081, R301082, R301084, and R301085 drug-dosing groups was terminated after D36; the detection of the R301079 and R301083 drug-dosing groups was terminated after D57.

[0217] Table 5 Changes in LPA protein levels in cynomolgus monkey serum after administration of siRNA conjugates

[0218]

[0219] The results showed that R301078, R301079, R301081, R301082, R301083, R301084, and R301085 could significantly reduce the LPA protein level in the serum of cynomolgus monkeys at a single dose of 3 mg / kg. The maximum inhibitory effect of R301078 on serum LPA protein was 97.20% (D29), and the protein reduction effect remained above 90% from D22 to D57 after administration; the protein level began to rise at D64 after administration, and the protein inhibition effect was still 78.26% at D92 (see Figure 1 , Table 5).

[0220] The above specific implementations are only illustrative of the content of the present invention and do not represent limitations of the content of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A double-stranded oligonucleotide for inhibiting LPA gene expression, wherein the double-stranded oligonucleotide is selected from RX301078, wherein the sense strand (5'-3') is CmsUmsGmGmCmUmUfGfAfUfCmAmAmGmAmAmCmUmAm; and the antisense strand (5'-3') is UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfGmCfCmAmGms(NM054)s(NM054); (NM054) represents a nucleotide in which the 2'-hydroxyl group and hydrogen of the ribose are replaced by [2'-F-2'-methyl], and its structural formula is ; C, G, U, A, and T represent cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, adenosine-3'-phosphate, and thymidine-3'-phosphate, respectively; m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl-modified nucleotide; f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorine-modified nucleotide; s represents that the two nucleotides before and after are connected by a thiophosphate backbone.

2. A conjugate, characterized in that The conjugate comprises the double-stranded oligonucleotide according to claim 1 and one or more ligands capable of binding to a cell surface receptor conjugated to the double-stranded oligonucleotide.

3. The conjugate according to claim 2, characterized in that The conjugate is selected from R301078, and the conjugate has a structure shown in the following formula (III) or a pharmaceutically acceptable salt thereof: Nu represents the double-stranded oligonucleotide according to claim 1.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the double-stranded oligonucleotide according to claim 1 or the conjugate according to any one of claims 2-3.

5. Use of the double-stranded oligonucleotide of claim 1, the conjugate of any one of claims 2-3, or the pharmaceutical composition of claim 4 in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene, wherein the disease or condition mediated by the LPA gene is a cardiovascular disease.

6. A kit, characterized in that: The kit comprises the double-stranded oligonucleotide according to claim 1, the conjugate according to any one of claims 2-3 or the pharmaceutical composition according to claim 4.

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