Double-stranded oligonucleotide inhibiting LPA gene expression and use thereof

Abstract

The present invention relates to the technical field of nucleic acid drugs. Disclosed are a double-stranded oligonucleotide inhibiting LPA gene expression and the use thereof. The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand contains a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the sequence as shown in SEQ ID NO. 1, and the antisense strand contains at least 17 nucleotides and is complementary or substantially complementary to the sense strand to form a duplex region. Each nucleotide of the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides. The double-stranded oligonucleotide can inhibit LPA gene expression and solve the problem of an siRNA drug for treating LPA gene expression related diseases such as cardiovascular and cerebrovascular diseases.

Description

Double-stranded oligonucleotides for inhibiting LPA gene expression and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application No. 202311061772.1 filed with the State Intellectual Property Office of China on August 22, 2023, entitled “Double-stranded oligonucleotides for inhibiting LPA gene expression and their applications,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] 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 applications thereof. Background Art

[0004] Lipoprotein (a) (Lp(a), LPA) is a low-density lipoprotein variant composed of a high-molecular-weight glycoprotein isoform homologous to plasminogen (PLG) and covalently bound to LDL-like particles via 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 primarily expressed in the liver and is limited to humans and non-primates. Plasma Lp(a) concentrations are largely determined by the LPA gene, which has played a key role in revealing the causal relationship between Lp(a) and cardiovascular disease. Lipoprotein (a) levels are a direct causal factor in a variety of cardiovascular diseases and are closely associated with 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 a cause of cardiovascular disease. Lp(a) is continuously associated with cardiovascular disease risk. Among 2,100 candidate genes for cardiovascular disease, variants in the LPA gene were found to be the strongest genetic risk factor for cardiovascular disease. Therefore, the etiological role of Lp(a) in cardiovascular disease has become a hot topic of research.

[0005] Small interfering RNA (siRNA), based on the RNA interference mechanism, 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, particularly cardiovascular diseases, can be prevented and treated at the cellular level. The guidelines for the prevention and treatment of adult dyslipidemia define Lp(a) abnormalities as >30 mg / d1. Based on this standard, approximately 30% of patients with a history of 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.

[0006] In view of this, the present disclosure is proposed.

[0007] Summary of the Invention

[0008] To address the needs of cardiovascular and cerebrovascular diseases, this disclosure develops a small nucleic acid drug targeting the LPA gene that can lower cholesterol levels and thereby prevent or control metabolic syndrome. A primary objective of this disclosure is to provide a double-stranded oligonucleotide that inhibits LPA gene expression, its conjugates, pharmaceutical compositions, and kits, as well as methods and uses thereof for inhibiting or reducing LPA gene expression or treating LPA-mediated diseases or symptoms.

[0009] In order to solve the above technical problems, the present disclosure adopts the following technical solutions:

[0010] In a first aspect, the present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand;

[0011] The positive strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the sequence shown in SEQ ID NO. 1, wherein the substantially identical nucleotide sequence has no more than 3 nucleotide differences from the at least 15 consecutive nucleotides;

[0012] wherein the antisense strand comprises at least 17 nucleotides and is complementary or substantially complementary to the sense strand to form a duplex region, wherein the substantially complementary region means that the sense strand and the antisense strand have no more than 3 nucleotide mismatches in the duplex region;

[0013] Each nucleotide of the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0014] In a second aspect, the present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression, wherein the double-stranded oligonucleotide comprises a sense strand and an antisense strand;

[0015] In the 5' to 3' direction, nucleotides 2-19 of the antisense strand comprise a region of complementarity to an LPA RNA transcript, wherein the complementary region comprises a sequence of at least 15, at least 16, at least 17, or at least 18 consecutive nucleotides of any one of SEQ ID NOs. 147-291, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides;

[0016] The sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand to form a duplex region; the substantial complementarity means that the mismatch between the sense strand and the antisense strand in the duplex region is no more than 3 nucleotides.

[0017] In a third aspect, the present disclosure provides a conjugate comprising the double-stranded oligonucleotide described above and one or more ligands capable of binding to a cell receptor.

[0018] In a fourth aspect, the present disclosure further provides use of the aforementioned double-stranded oligonucleotide or the aforementioned conjugate in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene.

[0019] In a fifth aspect, the present disclosure further provides a pharmaceutical composition comprising the aforementioned double-stranded oligonucleotide and / or the aforementioned conjugate.

[0020] In a sixth aspect, the present disclosure further provides a kit comprising the aforementioned double-stranded oligonucleotide, and / or the aforementioned conjugate, and / or the aforementioned pharmaceutical composition.

[0021] In a seventh aspect, the present disclosure further provides a method for inhibiting LPA gene expression, comprising administering the aforementioned double-stranded oligonucleotide, and / or the aforementioned conjugate, and / or the aforementioned pharmaceutical composition to a subject.

[0022] In an eighth aspect, the present disclosure further provides a method for alleviating, treating and / or preventing a disease or condition mediated by the LPA gene, the method comprising administering the aforementioned double-stranded oligonucleotide, and / or the aforementioned conjugate, and / or the aforementioned pharmaceutical composition to a subject.

[0023] Compared with the prior art, the present disclosure has the following beneficial effects:

[0024] The double-stranded oligonucleotides and pharmaceutical compositions provided by the present disclosure showed excellent LPA gene expression regulation activity in in vitro cell experiments and mouse model experiments. For example, the double-stranded oligonucleotides disclosed in the present disclosure showed excellent LPA mRNA inhibitory activity in LPA transgenic cell lines. At a siRNA concentration of 1 nM, the residual activity of the target gene LPA can be as low as 11.90% (RX001010). In the BALB / c mouse hydrodynamic injection model, the conjugates after siRNA conjugation with ligands can reduce the residual activity of the target gene LPA to 10.05% (RZ001005, dosage 3 mg / kg) and 18.76% (RZ001031, dosage 1 mg / kg). This shows that the double-stranded oligonucleotides, pharmaceutical compositions and oligonucleotide conjugates provided by the present disclosure can effectively regulate the expression level of the target gene LPA, and therefore can effectively treat and / or prevent disease symptoms related to the mRNA level expressed by the LPA gene, and have good application prospects.

[0025] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] FIG1 shows the inhibitory activity of target genes in BALB / c-HDI mice after administration of siRNA conjugates in Example 4 of the present disclosure;

[0028] FIG2 is a typical histopathological image of each test group at 200 times the field of view of HE staining in Example 5 of the present disclosure;

[0029] FIG3 shows the inhibitory activity of target genes in BALB / c-HDI mice after administration of siRNA conjugates in Example 6 of the present disclosure;

[0030] FIG4 shows the changes in the levels of Apo(a) protein in transgenic mice after administration of siRNA conjugates in Example 8 of the present disclosure.

[0031] FIG5 shows the changes in LPA protein levels in cynomolgus monkey serum after administration of siRNA conjugates in Example 9 of the present disclosure. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions of the present disclosure in conjunction with the embodiments. Obviously, the embodiments described are only a part of the embodiments of the present disclosure, not all of them. Based on the embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present disclosure.

[0033] [definition]

[0034] In this document, when letters refer to an object or quantity, they are case-sensitive. For example, Y and y represent different objects or quantities.

[0035] As used herein, a "nucleotide difference" between one nucleotide sequence and another nucleic acid sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleic acid sequences are considered to have a nucleic acid difference at that position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleic acid difference at that position can also be considered.

[0036] In this article, unless otherwise specified, the capital letters A (adenine), U (uracil), G (guanine), C (cytosine), and T (thymine) represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; (moe) indicates that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond.

[0037] Herein, the structural formula of nucleotide is

[0038] Herein, the structural formula of morpholino nucleic acid is

[0039] Wherein, 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.

[0040] Herein, the structural formula of the abasic nucleotide is

[0041] Herein, "2'-halogenated modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group of the nucleotide is replaced by a halogen atom. For example, a 2'-fluoro modified nucleotide.

[0042] Herein, "2'-O-optionally substituted C1-C6 alkyl modified nucleotides" refer to nucleotides in which the hydrogen atom of the 2' hydroxyl group of the ribose group of the nucleotide is replaced by an optionally substituted C1-C6 alkyl group. Exemplary are 2'-O-methyl (also known as 2'-methoxy) modified nucleotides.

[0043] Herein, "2'-deoxy-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a hydrogen atom.

[0044] In this article, "2'-O-(CH2) n -R1 modified nucleotides refer to nucleotides in which the hydrogen atom on the 2' hydroxyl group of the ribose group is replaced by -(CH2) n -R1 replaces the nucleotide formed; illustratively, when n is selected from 1, 2'-O-(CH2) n-R1 can be selected from 2'-O-CH2-O-CH2-CH3 (2'-O-ethoxymethyl) modified nucleotides or 2'-O-CH2-O-CH2-CF3 (also known as 2'-O-(2,2,2-trifluoroethoxymethyl)) modified nucleotides; when n is selected from 2, 2'-O-(CH2) n -R1 can be selected from 2'-O-CH2-CH2-O-CH3 (also known as 2'-O-methoxyethyl, 2'-O-MOE) modified nucleotides. Exemplarily, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0045] Herein, "2'-O-Si(R2)3 modified nucleotide" refers to a nucleotide in which the hydrogen atom on the 2' hydroxyl group of the ribose group of the nucleotide is replaced by -Si(R4)3. For example, 2'-O-TBDMS modified nucleotide, 2'-O-TIPS modified nucleotide or 2'-O-TOM modified nucleotide; wherein the structural formula of TBDMS is The structural formula of TIPS is The structural formula of TOM is

[0046] Herein, a double-stranded oligonucleotide consists of two strands, wherein the strand that binds to the target sequence is called the antisense strand or guide strand, and the other strand is called the sense strand or passenger strand.

[0047] As used herein, the term "antisense strand" refers to the strand of a double-stranded oligonucleotide that includes a region that is fully or substantially complementary to a target sequence.

[0048] As used herein, the term "sense strand" refers to the strand of a double-stranded oligonucleotide that includes a region that is substantially complementary to a region that is the antisense strand as that term is defined herein.

[0049] As used herein, the term "region of complementarity" refers to the region on the antisense strand that is completely or substantially complementary to the target sequence. Where the region of complementarity is not completely complementary to the target sequence, mismatches may be located in internal or terminal regions of the molecule. As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions.

[0050] As used herein, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases of the other strand in a complementary manner.

[0051] The terms "apolipoprotein (a) gene," "Apo(a) gene," and "LPA" are used interchangeably herein. LPA includes, but is not limited to, human LPA, cynomolgus monkey LPA, mouse LPA, and rat LPA. Their amino acid and complete coding sequences, as well as their mRNA sequences, are readily available from publicly available databases. These sequences can be naturally occurring, synthetic, recombinant, or any combination thereof.

[0052] As used herein, a "target sequence" refers to the contiguous portion of the nucleic acid sequence of the mRNA molecule formed during LPA transcription, including mRNAs that are processed as the primary transcription product. The targeted portion of the target sequence should be sufficiently long to serve as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is within the protein-coding region of LPA.

[0053] As used herein, the term "inhibit" can be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition. 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. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from an untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0054] As used herein, the term "small interfering RNA (siRNA)" refers to a type of double-stranded RNA consisting of a sense strand and an antisense strand. siRNAs mediate the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway by forming a silencing complex. Specifically, siRNAs direct the specific degradation of mRNA sequences through a process known as RNA interference (RNAi), inhibiting the translation of mRNA into amino acids and protein.

[0055] As used herein, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence.

[0056] As used herein, the term "sense strand" (or trailing strand) refers to the iRNA strand that is substantially complementary to the antisense strand.

[0057] As used herein, the term "substantially complementary" refers to complete complementarity or at least partial complementarity, e.g., the antisense strand is completely complementary or at least partially complementary to the target sequence. In the case of partial complementarity, mismatches can exist within the interior or terminal regions of the molecule, wherein the most tolerated mismatches exist within the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the iRNA.

[0058] Unless otherwise specified, the "compound," "ligand," "nucleic acid-ligand conjugate," "siRNA conjugate," "nucleic acid," "conjugate," "chemical modification," "targeting ligand," "dsRNA," and "RNAi" of the present disclosure may independently exist in the form of a salt, a mixed salt, or a non-salt form (e.g., a free acid or a free base). When present in the form of a salt or a mixed salt, it may be a pharmaceutically acceptable salt.

[0059] As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0060] As used herein, the term "pharmaceutically acceptable acid addition salt" refers to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, bromates, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, oxalates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbates, salicylates, 4-aminosalicylates, and disulphonates. These salts can be prepared by methods known in the art.

[0061] As used herein, the term "pharmaceutically acceptable base addition salt" refers to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, sylvite salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. These salts can be prepared by methods known in the art.

[0062] As used herein, the term "effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the course of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various conditions associated with the disclosed target genes, target mRNAs, or target proteins, or ameliorating one or more symptoms of the condition, reducing the dose of another agent required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a condition associated with the disclosed target genes, target mRNAs, or target proteins in a patient.

[0063] As used herein, the terms "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0064] As used herein, the terms "treat," "treat," or "alleviate" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. As used herein, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

[0065] As used herein, the terms "prevent" and "prevent" are used interchangeably to refer to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," a conjugate, RNAi agent, or composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0066] The double-stranded oligonucleotides provided in the present disclosure can be obtained by conventional preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. The modified nucleotide groups can be introduced into the double-stranded oligonucleotides described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.

[0067] As used herein, the term "chemical modification" or "modification" includes all changes in nucleic acids by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0068] As used herein, the term "base" encompasses any known DNA and RNA base, base analogues such as purine or pyrimidine, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, hypoxanthine, and natural analogues.

[0069] As used herein, unless otherwise stated, "optionally," "optional," "optional," or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0070] As used herein, the terms "comprise" or "include" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. As used herein, unless otherwise indicated, "comprising" or "including" encompasses the situation consisting of the stated elements, integers, or steps. For example, reference to "comprising" a particular sequence is intended to encompass the situation consisting of that particular sequence.

[0071] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2.3-dimethylpentyl, 2.4-dimethylpentyl, 2.2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4.4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylhexyl, n-decyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl and its various branched chain isomers, etc. In some embodiments, the group selected from the group consisting of alkyl groups having 1 to 6 carbon atoms includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. By way of example only, "C1-4 alkyl" means having one to four carbon atoms in the alkyl chain, i.e., the C1-4 alkyl is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl. Alkyl groups may be substituted or unsubstituted.

[0072] As used herein, the term "alkoxy" refers to an -O-alkyl group, wherein alkyl is as defined above. Non-limiting examples of such alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. By way of example only, "C 1-10 "Alkylene" means an alkyl group having one to ten carbon atoms in the alkyl chain. 1-10The alkylene group is selected from methylene (-CH2-), ethylene (=CH2CH3), 1,2-ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), and the like.

[0073] As used herein, the term "hydroxyl" refers to an -OH group.

[0074] As used herein, the term "halogen" or "halo" refers to any radioactive-stable atom of Group VII of the Periodic Table of Elements, such as fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred, and fluorine being more preferred.

[0075] As used herein, the term "connection" when referring to a connection between two molecules refers to the connection of two molecules by a covalent bond or the association of two molecules via a non-covalent bond (e.g., a hydrogen bond or an ionic bond), including direct connection and indirect connection. The term "direct connection" refers to the connection of a first compound or group to a second compound or group without any intervening atoms or groups of atoms. The term "indirect connection" refers to the connection of a first compound or group to a second compound or group via an intermediate group, compound, or molecule (e.g., a linking group).

[0076] As used herein, the term "optionally substituted" is used to define a variable that can be unsubstituted or substituted.

[0077] As used herein, the term "unsubstituted" means that the designated group bears no substituents.

[0078] As used herein, the terms "substituted," "substituted," and "substituted" are used interchangeably to indicate that any one or more hydrogen atoms in a given structure are replaced by a specified substituent (e.g., C 1-3 Alkyl, C 1-3 alkoxy or halogen), provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. Unless otherwise indicated, a substituted group may have a substituent at each substitutable position of the group. When more than one position in a given structural formula can be substituted with one or more substituents selected from a specified group, the substituents may be the same or different at each substitutable position.

[0079] Herein, the terms "each... is independently selected from" and "... are each independently selected from" and "... are independently selected from" are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0080] In the structural formulas of "compound", "ligand" and "support", the bond Indicates that the configuration is not specified. If chiral isomers exist in the chemical structure, the bond Can be or include both Although all of the above structural formulae are drawn as certain isomers for simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates, and enantiomers.

[0081] As used herein, the term "stereoisomers" refers to compounds that have identical chemical constitution but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like.

[0082] As used herein, the term "chiral" refers to a molecule that is non-superimposable on its mirror image, while "achiral" refers to a molecule that is superimposable on its mirror image.

[0083] As used herein, the term "enantiomers" refers to two non-superimposable isomers of a compound that are mirror images of each other.

[0084] As used herein, the term "diastereoisomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Diastereomeric mixtures can be separated by high-resolution analytical procedures such as electrophoresis and chromatography, for example, HPLC.

[0085] Herein, stereochemical definitions and conventions used generally follow those in SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994.

[0086] As used herein, the term "conjugation" refers to the covalent attachment of two or more chemical moieties, each with a specific function, to one another; accordingly, a "conjugate" refers to a compound formed by covalent attachment of two or more chemical moieties. Furthermore, a "drug conjugate" refers to a compound formed by covalent attachment of one or more chemical moieties with specific functions to an active drug. Sometimes, and especially in the examples, the "drug conjugate" of the present disclosure is also referred to as a "conjugate," "double-stranded oligonucleotide conjugate," or "siRNA conjugate." Drug conjugate should be understood as a general term for drug conjugates or a specific drug conjugate represented by a specific structural formula, depending on the context.

[0087] In a first aspect, the present disclosure provides a double-stranded oligonucleotide for inhibiting LPA gene expression. The double-stranded oligonucleotide is capable of inhibiting LPA gene expression. The double-stranded oligonucleotide can inhibit LPA gene expression in cells in vitro and in vivo.

[0088] The double-stranded oligonucleotide includes a sense strand and an antisense strand.

[0089] The positive strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the sequence shown in SEQ ID NO. 1, wherein the substantially identical means that the nucleotide sequence has no more than 3 (e.g., 0, 1, 2, or 3) nucleotide differences from the at least 15 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutive nucleotides.

[0090] In an optional embodiment, the positive strand comprises 15, 16, 17, 18, 19 or 20 consecutive nucleotides in the SEQ ID NO.1 sequence or a nucleotide sequence having no more than 2 (e.g., 0, 1 or 2) nucleotide differences with the consecutive nucleotides.

[0091] The antisense strand comprises at least 17 (e.g., 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28) nucleotides and is complementary or substantially complementary to the sense strand to form a duplex region, wherein the substantial complementarity means that the sense strand and the antisense strand have no more than 3 (e.g., 0, 1, 2, or 3) nucleotide mismatches in the duplex region.

[0092] In an optional embodiment, the antisense strand is complementary or substantially complementary to 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the nucleotide sequence of SEQ ID NO.1, and the substantially complementary means that the mismatch in the complementary region does not exceed 3 (e.g., 0, 1, 2 or 3) nucleotides.

[0093] In alternative embodiments, the duplex region of the double-stranded oligonucleotide is 15-25, 15-23, 15-21, 15, 19, 15-17, 16-25, 16-24, 16-22, 16-20, 16-18, 17-25, 17-23, 17-22, 17-20, 17-19, 18-25, 18-24, 18-23, 18-21, 18-20, 19-25, 19-23, 19-21, 20-25, 20-24, 20-22, 21-25, 21-23, 22-25, 22-24, or 23-25 ​​nucleotide pairs in length. The length of the duplex region of the double-stranded oligonucleotide is more specifically, for example but not limited to, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs.

[0094] In an alternative embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0095] In an alternative embodiment, substantially all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides. Here, "substantially all nucleotides in the double-stranded oligonucleotide are 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.

[0096] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotides, wherein n is selected from 1 or 2, and R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy. If R1 contains a substituent, the substitution in R1 refers to the replacement of one or more hydrogen atoms on the C1-C6 alkoxy or C1-C6 alkoxy with a substituent, and the substituent in R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy, or halogen.

[0097] In an optional embodiment, the 2'-O-(CH2) nThe -O-R1 modified nucleotide is selected from a 2'-O-methoxyethyl modified nucleotide, a 2'-O-ethoxymethyl modified nucleotide or a 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide.

[0098] In an optional embodiment, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0099] In an optional embodiment, the double-oligonucleotide is selected from siRNA.

[0100] In a second 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;

[0101] From the 5' end to the 3' end, nucleotides 2 to 19 of the antisense strand comprise a region of complementarity to an LPA RNA transcript, wherein the region of complementarity comprises a sequence of at least 15, at least 16, at least 17, or at least 18 consecutive nucleotides of any one of SEQ ID NOs. 147 to 291, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2, or 3 nucleotides.

[0102] From the 5' end to the 3' end, the sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand 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 1, 2 or 3 nucleotides.

[0103] In an optional embodiment, the nucleotide sequence of the antisense strand comprises 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 sequence in SEQ ID NO.147-291, or a nucleotide sequence with 1, 2 or 3 nucleotide differences from the consecutive nucleotides; in an optional embodiment, the antisense strand comprises a nucleotide sequence with 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NO.147-291; in an optional embodiment, the antisense strand comprises a nucleotide sequence with 0, 1 or 2 nucleotide differences from any one of SEQ ID NO.147-291; in an optional embodiment, the antisense strand comprises a nucleotide sequence with 0 or 1 nucleotide difference from any one of SEQ ID NO.147-291; in an optional embodiment, the antisense strand is selected from or comprises any one of SEQ ID NO.147-291.

[0104] In an optional embodiment, the nucleotide sequence of the sense strand comprises 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. 2-146, or has 1, 2 or 3 nucleotide differences from the consecutive nucleotides.

[0105] In an optional embodiment, the nucleotide sequence of the sense strand has 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 2-146.

[0106] In an optional embodiment, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0, 1 or 2 nucleotide differences from any one of SEQ ID NOs. 2-146.

[0107] In an optional embodiment, the nucleotide sequence of the sense chain comprises a nucleotide sequence having 0 or 1 nucleotide difference with any one of SEQ ID NO.2-146; in an optional embodiment, the nucleotide sequence of the sense chain is selected from or comprises any one of SEQ ID NO.2-146.

[0108] In an optional embodiment, the double-stranded oligonucleotide is selected from the modified or unmodified double-stranded oligonucleotide shown in any number RN001001-RN001145 in Table 1.

[0109] In an optional embodiment, the double-stranded oligonucleotide is selected from one or more of the modified or unmodified double-stranded oligonucleotides shown in the following numbers: RN001008, RN001010, RN001011, RN001025, RN001042, RN001074 and RN001121.

[0110] Table 1

[0111] In an alternative embodiment, each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides.

[0112] In an alternative embodiment, all nucleotides in the double-stranded oligonucleotide are substantially selected from modified nucleotides. Here, "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.

[0113] In an alternative embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides.

[0114] In an optional embodiment, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides:

[0115] 2'-halogenated modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-optionally substituted C1-C6 alkyl modified nucleotides, 2'-O-(CH2) n The invention also provides a nucleotide modified with 2'-O-R1, a 2'-O-Si(R2)3, a 2'-amino modified nucleotide, an abasic nucleotide, or a quasi-nucleotide; the quasi-nucleotide is selected from one or more of peptide nucleic acid (PNA), morpholino nucleic acid (morpholino nucleic acid), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid / glycerol nucleic acid (GNA), threose nucleic acid (TNA), and unlocked nucleic acid (UNA).

[0116] If the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide contains a substituent, the substitution in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide refers to the replacement of one or more hydrogen atoms in the C1-C6 alkyl group by a substituent, and the substituent in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0117] In an optional embodiment, the 2'-halogenated modified nucleotide is, for example but not limited to, selected from 2'-fluoro-modified nucleotides.

[0118] In an optional embodiment, the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is, for example but not limited to, selected from 2'-O-methyl modified nucleotides.

[0119] In an optional embodiment, the 2'-O-(CH2) n -O-R1 modified nucleotides, n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy;

[0120] If R1 contains a substituent, the substitution in R1 refers to a C1-C6 alkyl group or one or more hydrogen atoms in a C1-C6 alkyl group being replaced by a substituent, and the substituent in R1 is independently selected from a C1-C3 alkyl group, a C1-C3 alkoxy group or a halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0121] In an optional embodiment, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from a 2'-O-methoxyethyl modified nucleotide, a 2'-O-ethoxymethyl modified nucleotide or a 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide.

[0122] In an optional embodiment, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

[0123] In an optional embodiment, each R2 in the 2'-O-Si(R2)3 modified nucleotide is independently selected from unsubstituted or substituted C1-C6 alkyl;

[0124] If R2 contains a substituent, the substitution in R2 means that one or more hydrogen atoms in the C1-C6 alkyl group are replaced by a substituent, and the substituent in R2 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0125] In an optional embodiment, the 2'-O-Si(R2)3 modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide.

[0126] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotide or 2'-O-Si(R2)3 modified nucleotide.

[0127] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotides, such as 2'-O-methoxyethyl modified nucleotides.

[0128] In an optional embodiment, the antisense strand contains at least one 2'-O-(CH2) n -O-R1 modified nucleotides, such as 2'-O-methoxyethyl modified nucleotides.

[0129] In an optional embodiment, the sense strand contains at most two 2'-O-(CH2) n -O-R1 modified nucleotides, such as 2'-O-methoxyethyl modified nucleotides.

[0130] In an optional embodiment, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methyl modified nucleotides or 2'-O-methoxyethyl modified nucleotides.

[0131] In an alternative embodiment, the sense strand or the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0132] In an alternative embodiment, the antisense strand comprises a 3' overhang of at least 1 nucleotide.

[0133] In an alternative embodiment, the sense strand or the antisense strand comprises a 3' overhang of at least 2 nucleotides.

[0134] In an alternative embodiment, the antisense strand comprises a 3' overhang of at least 2 nucleotides.

[0135] In an optional embodiment, the sense strand is 18, 19, 20 or 21 nt in length, and the antisense strand is 19, 20, 21, 22 or 23 nt in length.

[0136] In an optional embodiment, the sense strand is 19 nt in length and the antisense strand is 21 nt in length.

[0137] In an alternative embodiment, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds.

[0138] In an alternative embodiment, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end.

[0139] In an alternative embodiment, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

[0140] In an optional embodiment, the structure of the duplex region of the double-stranded oligonucleotide is as shown in formula (101):

[0141] SS: 5'-(N)a'-(X)i'-(N)b'-(X)t'-(N)c'-(X)r'-(N)d'-3'

[0142] AS: 3'-(N)a-(X)i-(N)b-(X)t-(N)c-5' formula (101);

[0143] Wherein, SS represents the sense strand, and AS represents the antisense strand;

[0144] Each N is independently selected from a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, or a 2'-deoxy-modified nucleotide;

[0145] Each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides or 2'-O-Si(R2)3 modified nucleotides; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; each R2 is independently selected from optionally substituted C1-C6 alkyl; in an optional embodiment, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotide. In an optional embodiment, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide.

[0146] a, a', i, i', b, b', t, t', c, c', r', and d' independently represent the number of nucleotides; wherein a' is selected from an integer of 3-8, i' is selected from an integer of 0-3, b' is selected from an integer of 4-13, t' is selected from an integer of 0-4, c' is selected from an integer of 3-9, r' is selected from an integer of 0-3, d' is selected from an integer of 0-9, a is selected from an integer of 4-7, i is selected from an integer of 0-1, b is selected from an integer of 4-8, t is selected from an integer of 0-4, and c is selected from an integer of 6-10; and i', t', r', i, and t are not all 0 at the same time, and 0≤t'+r'≤4. In an optional embodiment, a' is selected from an integer of 3-8, i' is selected from 0 or 1, b' is selected from an integer of 4-13, t' is selected from 0 or 1, and c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, i is selected from 1, b is selected from an integer of 4-8, t is selected from 0 or 1, and c is selected from an integer of 6-10.

[0147] In an alternative embodiment, each N is independently selected from a 2'-fluoro modified nucleotide or a 2'-O-methyl modified nucleotide.

[0148] In an alternative embodiment, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide.

[0149] In an alternative embodiment, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide.

[0150] In an alternative embodiment, the duplex region represented by formula (101) comprises at least one 2'-O-methoxyethyl modified nucleotide or at least one 2'-O-ethoxymethyl modified nucleotide.

[0151] In an optional embodiment, the sense chain length of the double-stranded oligonucleotide is 18-21, 18-20, 18-19, 19-21, 19-20 or 20-21 nucleotides; more specifically, the sense chain length of the double-stranded oligonucleotide having the duplex region shown in formula (101) is 17, 18, 19, 20 or 21 nucleotides.

[0152] In an optional embodiment, the antisense strand length of the double-stranded oligonucleotide is 19-23, 19-22, 19-21, 19-20, 20-23, 20-22, 20-21, 21-23, 21-22 or 22-23 nucleotides; more specifically, the antisense strand length of the double-stranded oligonucleotide is 19, 20, 21, 22 or 23 nucleotides.

[0153] In an optional embodiment, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long.

[0154] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense chain 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides.

[0155] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7-10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, at most two nucleotides among the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence in the antisense chain 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0156] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, at most two nucleotides among the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense chain are selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0157] In an optional embodiment, in the direction from the 5' end to the 3' end, at least three nucleotides among the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense chain 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0158] In an optional embodiment, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense chain are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence in the antisense chain 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0159] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from a modified nucleotide, wherein the modification of the sense strand and the antisense strand is selected from any one of the following (1)-(10):

[0160] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 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; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0161] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 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; the nucleotides at positions 2, 6, 11, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0162] (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0163] (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0164] (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 10, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0165] (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 11, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0166] (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 12, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0167] (8) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 9 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide.

[0168] (9) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide.

[0169] (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, the nucleotides at positions 12 and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; the nucleotides at positions 2, 6, 11, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0170] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from:

[0171] In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0172] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from:

[0173] From the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 12, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides.

[0174] In an optional embodiment, the sense strand comprises one or two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end in the direction from the 5' end to the 3' end.

[0175] In an optional embodiment, the 5' end and the 3' end of the antisense strand independently contain 1 or 2 consecutive phosphorothioate bonds.

[0176] In an optional embodiment, in the direction from 5' end to 3' end, the terminal nucleotides at the 5' end of the sense strand contain 1 or 2 consecutive phosphorothioate bonds, and the 5' end and 3' end of the antisense strand each independently contain 1 or 2 consecutive phosphorothioate bonds.

[0177] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from a modified nucleotide, and the sense strand is selected from any one of A1-A11 among the sense strands listed in Table 2; and / or, the antisense strand is selected from any one of the antisense strands B1-B41 listed in Table 2.

[0178] In an optional embodiment, the double-stranded oligonucleotide is selected from any one of RX001149-RX001192 in Table 2:

[0179] Table 2

[0180] In an optional embodiment, the double-stranded oligonucleotide comprises a sense strand and an antisense strand; nucleotides 2-19 of the antisense strand comprise a complementary region to the LPA RNA transcript in the direction from the 5' end to the 3' end, wherein the complementary region comprises a sequence of at least 15, at least 16, at least 17 or at least 18 consecutive nucleotides of any one of SEQ ID NOs. 167, 188, 196, 202, 215, 220, 245 or 285, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2 or 3 nucleotides.

[0181] In an optional embodiment, the antisense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 167, 188, 196, 202, 215, 220, 245, and 285;

[0182] And / or, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEO ID NO. 22, 43, 51, 57, 70, 75, 100 or 140.

[0183] In an optional embodiment, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and the sense strand is selected from any one of A5, A8 or A12-A17 among the sense strands listed in Table 3; and / or, the antisense strand is selected from any one of the following B42-B49.

[0184] In an optional embodiment, the double-stranded oligonucleotide is selected from any one of R201078-R201085 in Table 3:

[0185] Table 3

[0186] Wherein, 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'-fluoro-modified nucleotide; the combination mark (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 phosphorothioate backbone;

[0187] (NM054) represents a nucleotide with the structural formula

[0188] In an optional embodiment, the double-oligonucleotide is selected from siRNA.

[0189] In the third aspect of the present disclosure, the present disclosure further provides a conjugate comprising the double-stranded oligonucleotide according to the first aspect or the double-stranded oligonucleotide according to the second aspect, and one or more ligands capable of binding to cell surface receptors.

[0190] In an optional embodiment, the 3' end and / or 5' end of the sense strand and / or the antisense strand is conjugated to the ligand.

[0191] In an optional embodiment, the 3' end and / or the 5' end of the sense strand is conjugated to the ligand.

[0192] In an alternative embodiment, the 3' end of the sense strand is conjugated to the ligand.

[0193] In an optional embodiment, the ligand is linked to the 3' end and / or 5' end of the sense strand and / or antisense strand via a phosphate group or a phosphorothioate group.

[0194] In an optional embodiment, the number of the ligands includes but is not limited to 1, 2, 3 or 4.

[0195] In an optional embodiment, the number of the ligand is 1.

[0196] In an optional embodiment, the number of the ligand is 1, and the ligand is conjugated to the 3' end of the sense strand.

[0197] In an alternative embodiment, the ligand is specifically targeted to liver tissue.

[0198] In an optional embodiment, the ligand is an asialoglycoprotein receptor ligand.

[0199] In an alternative embodiment, the ligand comprises galactose or a derivative thereof.

[0200] In an optional embodiment, the galactose derivative is N-acetylgalactosamine.

[0201] In an optional embodiment, the structure of the conjugate is shown in formula (201):

[0202] Wherein, Nu represents the double oligonucleotide, each R ligand are independently selected from ASGPR ligands, and y is selected from 1, 2, 3 or 4.

[0203] Each of the R ligand are independently conjugated to the 3' end and / or 5' end of the sense strand and / or the antisense strand, and each of the R ligand The conjugation positions are different.

[0204] In an optional embodiment, y is selected from 1 or 2.

[0205] In an optional embodiment, y is 1.

[0206] In an optional embodiment, y is 1, and the R ligand Conjugated to the 3' end of the sense strand.

[0207] In an optional embodiment, y is 2.

[0208] In an optional embodiment, y is 2, and the two R ligand conjugated to the 3' and 5' ends of the sense strand, respectively.

[0209] In an optional embodiment, each R ligand Each is independently selected from a molecule having a structure as shown in formula (301) or a pharmaceutically acceptable salt or stereoisomer thereof:

[0210] Wherein, * represents the conjugation site of the ligand on the sense strand or the antisense strand.

[0211] In formula (301), j is selected from 1, 2, 3 or 4, further optionally, j is selected from 1, 2 or 3; further optionally, j is 3.

[0212] In formula (301), each Z is independently selected from hydroxyl or thiol; further optionally, Z is hydroxyl.

[0213] In formula (301), each p is independently selected from 1, 2 or 3; further optionally, p is 1.

[0214] In formula (301), each q is independently selected from 1, 2 or 3; further optionally, q is 1.

[0215] In formula (301), each R3 is independently selected from H, an optionally substituted C1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group; if R3 contains a substituent, the substitution in R3 means that one or more hydrogen atoms in the C1-C6 alkyl group or the C1-C6 alkoxy group are replaced by a substituent, and the substituent in R3 is independently selected from C1-C3 alkyl group, C1-C3 alkoxy group or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0216] Further optionally, in formula (301), each R3 is H.

[0217] In formula (301), each L is independently selected from optionally substituted C1-C 20 Alkylene or R La and RLb are independently selected from optionally substituted C1-C 10 Alkylene, k is selected from 1, 2, 3, 4 or 5. If L contains a substituent, the substitution in L refers to C1-C 20 Alkylene or C1-C 10 One or more hydrogen atoms in the alkylene group are substituted by a substituent, and the substituent in L is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen (such as fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine, more preferably fluorine).

[0218] In an optional embodiment, each L is independently selected from C1-C 10 Alkylene or Among them, R La and R Lb are independently selected from C1-C5 alkylene, and k is 1, 2 or 3. Further optionally, k is 1.

[0219] In an optional embodiment, each L is independently selected from

[0220] In an optional embodiment, each L is independently selected from

[0221] In an optional embodiment, L is

[0222] In an optional embodiment, L is

[0223] In an alternative embodiment, each Y is independently selected from O, S or NH.

[0224] In an optional embodiment, Y is O.

[0225] In an optional embodiment, each R ligand Independently selected from molecules having a structure as shown in formula (302) or pharmaceutically acceptable salts or stereoisomers thereof:

[0226] In an optional embodiment, each R ligand Independently selected from molecules having any of the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

[0227] In an optional embodiment, each R ligand Independently selected from molecules having the structure shown below, or pharmaceutically acceptable salts thereof, or stereoisomers thereof:

[0228] In an optional embodiment, the conjugate is selected from a molecule having the structure shown in the following structural formula or a pharmaceutically acceptable salt or stereoisomer thereof:

[0229] in, represents the double-stranded oligonucleotide (eg, siRNA). Further, the ligand (CR01008×3) is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide via a phosphodiester bond.

[0230] In some other optional embodiments, each R ligand Independently selected from the following structures or pharmaceutically acceptable salts thereof:

[0231] In an optional embodiment, Nu in formula (201) is selected from any one of RX001149-RX001192 in Table 2.

[0232] In an optional embodiment, Nu is selected from any one of RX001149-RX001192 in Table 2, the ligand is selected from the formula (CR01008×3), y=1, and the ligand is conjugated to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0233] In an optional embodiment, the conjugate is selected from the double-stranded oligonucleotide conjugates shown by any number from RZ001031 to RZ001074 in Table 4.

[0234] Table 4

[0235] In the above table, it is explained exemplarily that the positive chain GmsGmsCmUmUmGmAfUfCfAfUmGmAmAmCmUmAmCmUm_(CR01008×3) in RZ001031 indicates that the ligand (CR01008×3) is conjugated to the 3' end of the sequence GmsGmsCmUmUmGmAfUfCfAfUmGmAmAmCmUmAmCmUm_(CR01008×3) as shown in SEQ ID NO.307.

[0236] In an optional embodiment, the conjugate is selected from the double-stranded oligonucleotide conjugates shown in any one of R301078-R301085 in Table 5.

[0237] Table 5

[0238] In a fourth aspect of the present disclosure, the present disclosure provides use of the double-stranded oligonucleotide of the first aspect, and / or the double-stranded oligonucleotide of the second aspect, and / or the conjugate of the third aspect in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene.

[0239] In a fifth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising the double-stranded oligonucleotide of the first aspect, and / or the double-stranded oligonucleotide of the second aspect, and / or the conjugate of the third aspect.

[0240] In the sixth aspect of the present disclosure, the present disclosure provides a kit comprising the double-stranded oligonucleotide of the first aspect, and / or the double-stranded oligonucleotide of the second aspect, and / or the conjugate of the third aspect, and / or the pharmaceutical composition of the fifth aspect.

[0241] In the seventh aspect of the present disclosure, the present disclosure provides a method for inhibiting LPA gene expression, the method comprising administering to a subject the double-stranded oligonucleotide of the first aspect, and / or the double-stranded oligonucleotide of the second aspect, and / or the conjugate of the third aspect, and / or the pharmaceutical composition of the fifth aspect.

[0242] In an eighth aspect of the present disclosure, the present disclosure provides a method for alleviating, treating and / or preventing a disease or condition mediated by the LPA gene, the method comprising administering to a subject the double-stranded oligonucleotide of the first aspect, and / or the double-stranded oligonucleotide of the second aspect, and / or the conjugate of the third aspect, and / or the pharmaceutical composition of the fifth aspect.

[0243] The LPA gene-mediated diseases or disorders in any of the above aspects include diseases associated with LPA gene expression.

[0244] In an alternative embodiment, the disease or condition mediated by the LPA gene comprises cardiovascular disease.

[0245] In an optional embodiment, the cardiovascular disease includes but is not limited to: hyperlipoprotein (a) blood, Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and / or any other disease or pathology associated with elevated levels of Lp (a) particles.

[0246] In an optional embodiment, the double-stranded oligonucleotide and / or the conjugate in any of the above embodiments; or the double-stranded oligonucleotide and / or the conjugate in the pharmaceutical composition are administered in an unbuffered solution or a buffer.

[0247] In alternative embodiments, the unbuffered solution is saline or water.

[0248] In an optional embodiment, the buffer component in the buffer comprises acetate, citrate, prolamin, carbonate or phosphate or any combination thereof.

[0249] In an alternative embodiment, the buffer is phosphate buffered saline (PBS).

[0250] In an optional embodiment, the subject of the double-stranded oligonucleotide and / or the conjugate in any of the above embodiments is a cell.

[0251] In an optional embodiment, the subject is a cell, and the method comprises substantially contacting the subject cell with the double-stranded oligonucleotide, and / or the conjugate, and / or the pharmaceutical composition.

[0252] In an optional embodiment, the subject is a cell, and the administration method comprises: (a) contacting the cell with the double-stranded oligonucleotide, and / or the conjugate, and / or the pharmaceutical composition; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the LPA gene, thereby inhibiting the expression of the LPA gene in the cell.

[0253] In an optional embodiment, the subject is a human.

[0254] In an optional embodiment, the subject is a cell derived from a human.

[0255] In an optional embodiment, the inhibition of LPA gene expression is inhibition of LPA gene expression by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70%. The inhibition rate of LPA gene expression can be determined by conventional methods known in the art, such as, but not limited to, luciferase reporter gene assay, PCR, or protein-based methods, such as immunofluorescence analysis, Western Blot, or flow cytometry.

[0256] In an optional embodiment, the double-stranded oligonucleotide and / or the conjugate is administered at a dose of 0.01 mg / kg to 10 mg / kg or 0.5 mg / kg to 50 mg / kg.

[0257] In an alternative embodiment, the double-stranded oligonucleotide and / or the conjugate is administered subcutaneously or intravenously.

[0258] The present disclosure is further illustrated below through specific examples. However, it should be understood that these examples are only used for more detailed description and should not be understood as limiting the present disclosure in any form.

[0259] Unless otherwise specified, the siRNA sequences used in the present disclosure were commissioned to Kunshan Aotai Biotechnology Co., Ltd. for synthesis; the synthesis of PCR primers used in the present disclosure was commissioned to Sangon Biotech (Shanghai) Co., Ltd.; the HEK293-LPA stable cell line used in the present disclosure was donated by Beijing Institute of Technology; the experimental animals BALB / c mice used in the present disclosure were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.; the experimental animals ICR mice used in the present disclosure were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.; the hApo(a) transgenic mice used in the present disclosure were constructed by Suzhou Xuanjing Biotechnology Co., Ltd. and commissioned to Saiye (Suzhou) Biotechnology Co., Ltd.; the serum samples of the transgenic mice in the present disclosure were detected using a human Lipoprotein A ELISA kit.

[0260] Unless otherwise stated, the real-time PCR detection data of the activity experiments involved in this disclosure all use the ΔΔCt method to perform relative quantitative calculation of the target gene mRNA in each test group. The calculation method is summarized as follows:

[0261] ΔCt(test group)=Ct(test group target gene)-Ct(test group internal reference gene);

[0262] ΔCt(control group)=Ct(control group target gene)-Ct(control group internal reference gene);

[0263] ΔΔCt(test group)=ΔCt(test group)-ΔCt(average of control group);

[0264] ΔΔCt(control group)=ΔCt(control group)-ΔCt(average of control group).

[0265] The mRNA expression level of the target gene in the test group was normalized with the control group as the benchmark, and the remaining expression level of the target gene mRNA in the control group was defined as 100%;

[0266] Relative residual expression level of target gene mRNA in the test group = 2 -ΔΔCt (test group) × 100%;

[0267] Target gene mRNA inhibition rate in the test group = 100% - relative expression level of target gene mRNA in the test group;

[0268] The calculation method for the detection data of Apo(a) protein at different time points in the in vivo activity experiment involved in the present disclosure is summarized as follows:

[0269] 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.

[0270] In the context of the present disclosure, unless otherwise stated, the reagent ratios provided below are calculated on a volume basis (v / v).

[0271] Preparation of compounds

[0272] Unless otherwise stated, all 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 6.

[0273] Table 6

[0274] Among them, CPG stands for controlled pore glass (Controlled Pore Glass) carrier.

[0275] Preparation Example 1 Preparation of Compound CR01008

[0276] (1.1) The synthetic structure of compound CR01008 is shown below:

[0277] The synthetic route of compound CR01008 is as follows:

[0278] (1.1.1) Synthesis of Compound 2

[0279] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 10.0 g, 1.0 eq) and formaldehyde solution (8.9 g, 37% by mass, 2.4 eq) were dissolved in 33 ml of methanol. 13 ml of a 45.3% by mass aqueous solution of KOH was added dropwise. After the addition was complete, the mixture was stirred at 25°C for 30 minutes, then heated 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 white solid. A small amount of water was added to the crude product, and the mixture was slurried and filtered to obtain compound 2 (9 g, 78.9% yield) as a white solid. MS-ESI (m / z) = 260 [M+H] + .

[0280] (1.1.2) Synthesis of compound 3

[0281] Compound 2 (9 g, 1 eq) prepared in step (1.1.1) was dissolved in 70 mL of 1,4-dioxane. A 4 M solution of hydrogen chloride in 1,4-dioxane (45 mL) was added, and the mixture was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (6.8 g, 100% yield) as a white solid.

[0282] (1.1.3) Synthesis of compound 5

[0283] Compound 3 (1.8 g, 2.0 eq), compound 4 (5-[[(2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)-2-tetrahydropyranyl]oxy]pentanoic acid, 2.1 g, 1.0 eq), and DIEA (N,N-diisopropylethylamine, 3.5 g, 6.0 eq) prepared in step (1.1.2) were dissolved in 15 ml of DMF. HBTU (1.9 g, 1.1 eq) was added, and the mixture was stirred at 25°C under a N2 atmosphere for 3 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (22% acetonitrile in water by volume) to obtain compound 5 (1.78 g, 64.4% yield) as a white solid. MS-ESI (m / z) = 589 [M+H] + .

[0284] (1.1.4) Synthesis of Compound 6

[0285] 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 using an ice-water bath and DMTrCl (4,4'-dimethoxytriphenylmethane chloride, 1.32 g, 1.5 eq) was added at 0°C. The reaction was allowed to react at 25°C for 3 hours. 15 ml of methanol was added to the reaction solution to quench the reaction. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (60% acetonitrile in water by volume) to obtain compound 6 (1 g, 42.7% yield) as a yellow solid. MS-ESI (m / z) = 891 [M+H] + .

[0286] (1.1.5) Synthesis of compound CR01008

[0287] Compound 6 (1.08 g, 1.0 eq) prepared in step (1.1.4) was dissolved in 20 ml of anhydrous dichloromethane. 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 purged with nitrogen three times, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, 20 ml of saturated aqueous sodium bicarbonate 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 column chromatography (72 vol% acetonitrile in water) and then dried under vacuum for 12 hours to obtain compound CR01008 (1 g, 76.0% yield) as a white powder. MS-ESI (m / z) = 1091 [M+Na] + .

[0288] H NMR(400MHz, DMSO-d6)δ1.05(d,J=6.7Hz,6H).1.14(d,J=6.7Hz,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=5.9Hz,2H),2.79(d,J=8.4Hz,1H),2.87(d,J=8.4Hz,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=11.2,8.8Hz,1H),4.05(s,3H),4.51(d,J=8.4Hz,1H),4.99(dd,J=11.3,3.4Hz,1H) ,5.24(d,J=3.4Hz,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=7.8Hz,1H),7.84(d,J=9.2Hz,1H).

[0289] (1.2) Synthesis of compound CR01008Z

[0290] The synthetic route of compound CR01008Z is as follows:

[0291] (1.2.1) Synthesis of compound 9

[0292] 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 atmosphere was purged with nitrogen three times, and the mixture was stirred at 25°C for 16 hours. Flash purification was performed to obtain compound 9 (300 mg, yield 53.6%). MS-ESI (m / z) = 1013 [M+Na] + .

[0293] (1.2.2) Synthesis of compound CR01008Z

[0294] 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 vial and shaken for 16 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo. 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 vial and shaken for 6 hours. After the reaction, the reaction solution was filtered to obtain a filter cake, which was washed once with 10 ml of acetonitrile (1×10 ml) and then dried in vacuo to obtain compound CR01008Z (1.03 g, loading 20-30 μmol / g).

[0295] Among them, Cap1 and Cap2 are capping reagents, Cap1 is a pyridine / acetonitrile mixed solution of N-methylimidazole with a concentration of 20 volume%, and the volume ratio of pyridine to acetonitrile is 3:5; Cap2 is an acetonitrile solution of acetic anhydride with a concentration of 20 volume%.

[0296] Preparation Example 2 Preparation of Compound CR01013

[0297] (2.1) Synthesis of compound CR01013

[0298] The synthetic route of compound CR01013 is as follows:

[0299] (2.1.1) Synthesis of compound 2

[0300] Compound 1 (trans-4-(Boc-amino)cyclohexylcarboxaldehyde, 4.9 g) was dissolved in 17 ml of methanol, and aqueous formaldehyde solution (4.21 g, concentration of 37% by mass) and aqueous sodium hydroxide solution (6.5 ml, concentration of 45.3% by mass) were added dropwise. After the addition was complete, the temperature was raised to 60°C and stirred at 60°C for 2 hours. After the reaction was completed, the reaction solution was cooled to 25°C and evaporated to dryness under reduced pressure to obtain a crude white solid. A small amount of water was added to the crude product to make a pulp, filtered and dried to obtain compound 2 (4.8 g, yield 85.9%) as a white solid. ESI-MS (m / z) = 260.2 [M+H] + .

[0301] (2.1.2) Synthesis of compound 3

[0302] Compound 2 (4.8 g), prepared in step (2.1.1), was dissolved in 25 ml of 1,4-dioxane. A 4 M solution of hydrochloric acid in 1,4-dioxane (25 ml) was added, and the mixture was stirred at 25°C for 2 hours. After completion of the reaction, the reaction solution was evaporated to dryness under reduced pressure to obtain compound 3 (3.6 g, 99.4% yield) as a white solid.

[0303] (2.1.3) Synthesis of compound 11

[0304] Compound 3 (3.6 g) prepared in step (2.1.2) was dissolved in 36 ml of DMF, and TEA (5.62 g), compound 10 (N-benzyloxycarbonyl-4-aminobutyric acid, 5.28 g), and HBTU (8.43 g) were added, and the mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 200 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 100 ml of ethyl acetate (3×100 ml). The organic phases were combined, washed once with 50 ml of saturated aqueous sodium chloride solution (1×50 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 10 / 1, v / v) to obtain compound 11 (2.3 g, yield 33.0%) as a white solid. ESI MS (m / z) = 379.5 [M+H] + .

[0305] (2.1.4) Synthesis of compound 12

[0306] Compound 11 (2.3 g) prepared in step (2.1.3) was dissolved in 23 ml of methanol, and wet palladium on carbon (230 mg, 10% loading) was added. The atmosphere was replaced with hydrogen three times, and the reaction system was stirred at 25°C under a hydrogen atmosphere (15 psi) for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filtrate, which was evaporated to dryness under reduced pressure to obtain compound 12 (1.48 g, 99.8% yield) as a yellow oil.

[0307] (2.1.5) Synthesis of compound 13

[0308] Compound 12 (1.48 g) prepared in step (2.1.4) was dissolved in 15 ml of DMF, and triethylamine (TEA, 1.22 g), compound 4 (1.35 g), and HBTU (3.45 g) were added. The mixture was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was added to 150 ml of saturated aqueous sodium bicarbonate solution and extracted three times with 50 ml of ethyl acetate (3 × 50 ml). The organic phases were combined, washed once with 30 ml of saturated aqueous sodium chloride solution (1 × 30 ml), and then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness under reduced pressure and purified by reverse phase column chromatography (C18 column, eluent: water / acetonitrile = 5 / 1, v / v) to obtain compound 13 (1.3 g, yield 31.8%) as a white solid. ESI-MS (m / z): 674.3 [M+H] + .

[0309] (2.1.6) Synthesis of Compound 14

[0310] Compound 13 (1.1 g) prepared in step (2.1.6) was dissolved in 11 ml of pyridine. The reaction system was cooled to 0°C using an ice-water bath and DMTrCl (813 mg) was added portionwise at 0°C. The reaction system was stirred at 0°C for 1 hour. After the reaction was completed, methanol was added to the reaction solution to quench the reaction. The solvent was evaporated and the solution was purified by reverse phase column chromatography (eluent: water / acetonitrile = 1 / 4, v / v) to obtain compound 14 (800 mg, yield 50.3%) as a white solid. ESI-MS (m / z): 976.5 [M+H] + .

[0311] (2.1.7) Synthesis of compound CR01013

[0312] At 25°C, compound 14 (550 mg) was dissolved in 5 ml of dichloromethane (DCM), and 4,5-dicyanoimidazole (DCl, 53.2 mg) and compound 7 (2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, 255.4 mg) were added. The atmosphere was purged with nitrogen three times, and the reaction system was stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was washed twice with 5 ml of saturated sodium bicarbonate solution (2 × 5 ml) and once with 30 ml of saturated sodium chloride solution (1 × 30 ml). The organic phase was separated and dried over anhydrous sodium sulfate. The organic phase solvent was evaporated to dryness under reduced pressure and purified by normal phase column chromatography (eluent: dichloromethane / methanol = 20 / 1, v / v) to obtain compound CR01013 (532 mg, yield 80.4%) as a white solid. ESI-MS (m / z): 1176.7 [M+H] + .

[0313] 1H NMR (400MHz, DMSO-d6) δ0.95-1.05 (d, J=6.7Hz, 5H), 1.06-1.15 (q, J=7.6Hz, 8H), 1.15-1.21 (t, J=7.2Hz, 14H), 1.72-1 .80(s,3H),1.84-1.92(s,3H),1.94-2.07(d,J=16.0Hz,7H),2.07-2.14(s,3H),2.64-2.72(q,J=5.8Hz,2H),2.74-2.89 (d,J=8.5Hz,2H),3.35-3.56(m,4H),3.57-3.70(m,4H),3.71-3.77(s,6H),3.81-3.93(m,1H),3.96-4.09(d,J=6.4Hz, 3H), 6.82-6.97 (d, J=8.7Hz, 4H), 7.17-7.27 (t, J=8.7Hz, 5H), 7.27-7.34 (t, J=7.6Hz, 2H), 7.34–7.43 (d, J=7.5Hz, 2H).

[0314] (2.2) Synthesis of compound CR01013Z

[0315] The synthetic route of compound CR01013Z is as follows:

[0316] (2.2.1) Synthesis of compound 15:

[0317] Compound 14 (100 mg, 0.10 mmol) was dissolved in 2 ml of dichloromethane at 25°C. Triethylamine (25.9 mg, 0.25 mmol), DMAP (1.25 mg, 0.01 mmol), and compound 8 (succinic anhydride, 15.4 mg, 0.15 mmol) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the solvent was evaporated and the product was purified by reverse-phase column chromatography (C18 column, eluent: water / acetonitrile = 2 / 1, v / v) to obtain compound 15 (110 mg, 0.10 mmol, 100% yield) as a yellow oil. ESI-MS (m / z) = 1099.3 [M+Na]+.

[0318] (2.2.2) Synthesis of compound CR01013Z:

[0319] Compound 15 (50 mg, 0.04 mmol) was dissolved in 10 ml of acetonitrile, and HBTU (24.2 mg, 0.06 mmol), DIEA (11.0 mg, 0.08 mmol), and amino-CPG (1.06 g, loading 80 μmol / g) were added. The reaction system was stirred at 25°C for 16 hours. After completion of the reaction, the reaction solution was filtered to obtain a filter cake, which was washed twice with 50 ml of dichloromethane (2 × 50 ml), three times with 50 ml of acetonitrile (3 × 50 ml), and once with 50 ml of ethyl acetate (1 × 50 ml), and then vacuum dried. Cap 1 (4.8 ml), Cap 2 (0.54 ml), and DMAP (2.59 mg) were added to the dried filter cake, and the reaction system was stirred at 25°C for 5 hours. After the reaction was completed, the reaction solution was filtered to obtain a filter cake, which was washed three times with 50 ml of acetonitrile (3×50 ml) and dried in vacuo to obtain compound CR01013Z (900 mg, loading amount of 20-30 μmol / g).

[0320] Cap1 and Cap2 are capping reagents. Cap1 is a 20% by volume N-methylimidazole mixed solution in pyridine / acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; Cap2 is a 20% by volume acetic anhydride solution in acetonitrile.

[0321] Preparation Example 3: Synthesis of Compound NM054

[0322] In this preparation example, the synthetic route of compound NM054 is as follows:

[0323] (3-1) Synthesis of Compound NM054-2

[0324] 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, and 4,4'-bismethoxytrityl chloride (4.29 g, 12.7 mmol, 1.1 eq) was added portionwise. The atmosphere was purged with nitrogen three times. The reaction system was stirred at 25°C under a nitrogen atmosphere for 3 hours. HPLC indicated the absence of starting material. After completion of the reaction, the reaction solution was concentrated and extracted with purified water (50 ml) and ethyl acetate (50 ml). The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to yield compound NM054-2 (2.7 g, 41.7% yield). MS ESI (m / z) = 563.0 [M+H] + .

[0325] (3-2) Synthesis of Compound NM054

[0326] To a 100 ml reactor was added compound NM054-2 (2.7 g, 4.8 mmol, 1.0 eq), followed by the addition of bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.74 g, 5.76 mmol, 1.2 eq) in portions. 4,5-dicyanoimidazole (0.45 g, 3.8 mmol, 0.8 eq, DCI, CAS No. 1122-28-7) and dichloromethane (27 ml). The atmosphere was purged with nitrogen three times, and the reaction system was stirred at 25°C under a nitrogen atmosphere for 3 hours. After completion of the reaction, aqueous sodium bicarbonate solution (20 ml) was added to the reaction solution, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (eluent: acetonitrile / water = 90 / 10, v / v) to afford compound NM054 (3.0 g). MS ESI (m / z) = 763 [M+H] + .

[0327] Preparation of siRNA / siRNA conjugates

[0328] Preparation Example 4: Preparation of siRNA

[0329] (4-1) Synthesis of the Sense Chain (SS)

[0330] By using the phosphoramidite nucleic acid solid phase synthesis method, the compound on the solid phase support is linked to nucleoside monomers one by one in the 3'-5' direction according to the nucleotide sequence. During the synthesis process, compound NM054 is regarded as a nucleoside monomer.

[0331] Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis conditions are given below:

[0332] The nucleoside monomer was prepared into an acetonitrile solution with a concentration of 0.1 M.

[0333] The deprotection reaction conditions for each step were identical: 25°C, 70 seconds, a 3% vol. dichloroacetic acid solution in dichloromethane as the deprotection reagent, and a 5:1 molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support.

[0334] The conditions for each coupling reaction were the same. The coupling reaction conditions were: temperature 25°C, a molar ratio of the nucleic acid sequence attached to the solid support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence attached to the solid support to the coupling reagent of 1:65, a reaction time of 600 seconds, a 0.5 M solution of 5-ethylthio-1H-tetrazole in acetonitrile as the coupling reagent, and a 0.2 M solution of hydrogenated xanthan gum in acetonitrile / pyridine (1:1 volume ratio of acetonitrile to pyridine) as the thiolation reagent.

[0335] The capping reaction conditions were identical for each step. The capping reaction conditions were: 25°C; 2 minutes; a 1:1 molar ratio of Cap1 and Cap2; Cap1: a 20% by volume N-methylimidazole solution in pyridine / acetonitrile (with a 3:5 volume ratio of pyridine to acetonitrile); and Cap2: a 20% by volume solution of acetic anhydride in acetonitrile. The molar ratio of the N-methylimidazole in Cap1 to the acetic anhydride in Cap2 to the nucleic acid sequence attached to the solid support was 1:1:1.

[0336] The oxidation reaction conditions were identical for each step. The oxidation reaction conditions were: temperature, 25°C; reaction time, 3 seconds; oxidizing agent concentration, 0.05 M iodine solution; a molar ratio of iodine to the nucleic acid sequence attached to the solid support during the coupling reaction, 30:1; and the oxidation reaction was performed in a water / pyridine mixture (1:9 by volume). The sulfidation reaction conditions were: temperature, 25°C; reaction time, 360 seconds; thiolation agent concentration, 0.2 M hydrogenated xanthan gum in pyridine solution; a molar ratio of thiolation agent to the nucleic acid sequence attached to the solid support during the coupling reaction, 4:1; and the sulfidation reaction was performed in a water / pyridine mixture (1:9 by volume).

[0337] After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase support is cut, deprotected, purified, desalted, and then freeze-dried to obtain the positive chain, wherein:

[0338] Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence attached to the solid support was added to 0.5 ml / μmol of 25% by mass ammonia water at 55°C for 16 hours. The solvent was then removed and the product was concentrated to dryness in vacuo. After the ammonia treatment, the product was dissolved in 0.4 ml / μmol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid. Subsequently, 0.3 ml / μmol of triethylamine and 0.6 ml / μmol of triethylamine trihydrofluoride were added to remove the 2'-O-TBDMS protection from the ribose.

[0339] Purification and desalting conditions: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with a NaCl gradient elution. Specifically, eluent 1 consisted of 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); eluent 2 consisted of 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) in a water / acetonitrile mixture (9:1 volume ratio of water to acetonitrile); the elution gradient was eluent 1:eluent 2 = (100:0) to (50:50). The product eluates were collected and combined, and desalted using a reversed-phase chromatography column. Desalting conditions included using a Sephadex column with Sephadex G25 as the filler and eluting with deionized water.

[0340] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC); molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value. If the measured value was consistent with the theoretical value, the positive strand of the siRNA was obtained.

[0341] (4-2) Synthesis of antisense strand (AS)

[0342] The antisense chain was synthesized using a universal solid phase support. 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 (4-1) for synthesizing the sense chain.

[0343] Detection: Purity is detected using ion exchange chromatography (IEX-HPLC); molecular weight is detected using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weight is compared with the theoretical value. If the measured value is consistent with the theoretical value, it indicates that the antisense strand of the siRNA has been obtained.

[0344] (4-3) siRNA Synthesis

[0345] The sense chain synthesized in step (4-1) and the antisense chain synthesized in step (4-2) were mixed in an equimolar ratio, dissolved in water for injection and heated to 95° C., slowly cooled to room temperature and maintained at room temperature for 10 minutes to allow the sense chain and antisense chain to form a double-stranded structure through hydrogen bonds, thereby obtaining siRNA having the sense chain and antisense chain shown in Table 7.

[0346] Table 7 siRNA sequence information

[0347] In the above table, the modified nucleotide sequence represented by the positive chain of RX000001 is the naked sequence represented by SEQ ID NO.305, and the modified nucleotide sequence represented by the antisense chain of RX000001 is the naked sequence represented by SEQ ID NO.306.

[0348] Preparation Example 5 Preparation of siRNA Conjugate with the 3' End of the Sense Strand Conjugated to the L96 Vector

[0349] Compound L96-PS was purchased from Asymchem Pharmaceuticals (Tianjin) Co., Ltd. with a loading of 120±12 μmol / g (detection method: UV / HPLC). The structural formula of compound L96-PS is shown below (R-L96-PS):

[0350] Wherein, PS represents polystyrene resin solid phase carrier.

[0351] (5-1) Synthesis of the positive chain

[0352] Using the phosphoramidite nucleic acid solid-phase synthesis method, the L96-PS compound attached to the solid support was used as the initial cycle, and nucleoside monomers were attached one by one in the 3'-5' direction according to the nucleotide sequence. Each nucleoside monomer attachment involved a four-step reaction: deprotection, coupling, capping, and oxidation or sulfurization. The synthesis method and conditions were similar to the sense strand synthesis method described in step (4-1) of Preparation Example 4.

[0353] (5-2) Synthesis of antisense strand

[0354] The antisense strand of the siRNA conjugate was synthesized according to the antisense strand synthesis method shown in step (4-2) of Preparation Example 4.

[0355] (5-3) Synthesis of siRNA conjugates

[0356] The siRNA conjugate in which the 3' end of the sense strand was conjugated to the L96 vector was synthesized according to the siRNA synthesis method described in step (4-3) of Preparation Example 4. The structural formula of the siRNA conjugate in which the 3' end of the sense strand was conjugated to the L96 vector is shown below:

[0357] in, L96 is conjugated to the 3' end of the sense strand of the siRNA via a phosphodiester bond.

[0358] Table 8 Sequence information of siRNA conjugates with the 3' end of the sense strand conjugated to the L96 vector

[0359] In the above table, the modified nucleotide sequences shown in the sense strands of RZ000001 and RZ000002 correspond to the naked sequence shown in SEQ ID NO.301, and the modified nucleotide sequences shown in the antisense strands of RZ000001 and RZ000002 correspond to the naked sequence shown in SEQ ID NO.302.

[0360] Explained illustratively, the positive chain UmsUmsCmUmCmCmGfAfAfCmGmUmGmUmCmAmCmGmUm_L96 in number RZ000001 indicates that the ligand L96 is conjugated to the 3' end of the sequence UmsUmsCmUmCmCmGfAfAfCmGmUmGmUmCmAmCmGmUm as shown in SEQ ID NO.648 through a phosphodiester bond.

[0361] Preparation Example 6 Synthesis of siRNA Conjugate Conjugated to the 3' End of the Sense Strand (CR01008×3) Vector

[0362] (6-1) Synthesis of the positive strand of the 3' end conjugated (CR01008×3) vector

[0363] By phosphoramidite nucleic acid solid phase synthesis method, starting with the compound CR01008Z attached to the solid phase support, nucleoside monomers are linked one by one in a 3'-5' direction according to the nucleotide sequence. During the synthesis process, compound CR01008 is regarded as a nucleoside monomer.

[0364] Each connection of a compound involves four steps: deprotection, coupling, capping, and oxidation or sulfurization. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection conditions, and purification and desalting conditions in the synthesis of the sense chain in this preparation example are the same as those in the synthesis of the sense chain in step (4-1) of Preparation Example 4.

[0365] During the synthesis of the positive chain, the present disclosure obtains three clusters of CR01008, which are denoted as (CR01008×3) or (CR01008)×3.

[0366] The structural formula of the three-cluster CR01008 is as follows:

[0367] (6-2) Synthesis of antisense strand

[0368] The antisense chain of this preparation example was synthesized according to the antisense chain synthesis method shown in step (4-2) of Preparation Example 4.

[0369] (6-3) siRNA conjugate with the 3' end of the sense chain conjugated to a (CR01008×3) carrier. The siRNA conjugate with the 3' end of the sense chain conjugated to a (CR01008×3) carrier was synthesized according to the method described in step (4-3) of Preparation Example 4.

[0370] The structural formula of the siRNA conjugate with the 3' end of the sense strand conjugated to the (CR01008×3) carrier is as follows:

[0371] in, Represents siRNA. (CR01008×3) is conjugated to the 3' end of the sense strand of the siRNA via a phosphodiester bond.

[0372] Table 9 Sequence information of siRNA conjugates with the 3' end of the positive strand conjugated to a vector (CR01008×3)

[0373] In the sequences disclosed herein, the base composition and modification meanings are as follows: capital letters A, U, G, C, and T represent the base composition of the nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-O-methyl-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide; lowercase letter d represents that the nucleotide adjacent to the left of the letter d is a 2'-deoxy-modified nucleotide (also known as deoxynucleotide); lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate bond; and the combination mark (moe) represents that the nucleotide adjacent to the left of the combination mark (moe) is a 2'-O-methoxyethyl-modified nucleotide.

[0374] (NM054) represents a nucleotide with the structural formula

[0375] Biological detection experiments

[0376] Unless otherwise stated, the reagents, consumables, and instruments used in the biological detection experiments of this disclosure are all commercially available products. Among them, the main reagents and consumables and their sources are shown in Table 10, and the main instruments and their sources are shown in Table 11.

[0377] Table 10 Main reagents and consumables

[0378] Table 11 Main instruments and equipment

[0379] Example 1 Construction of LPA transgenic cell line:

[0380] (1.1) Construction of LPA expression plasmid

[0381] The human LPA gene mRNA sequence (NM_005577.4) was obtained from NCBI and fully synthesized by Suzhou GeneWeiZhi Biotechnology Co., Ltd. The gene was then inserted into the multiple cloning site of the pcDNA3.1 vector to construct the pcDNA3.1-hLPA expression vector. Plasmid amplification, extraction, and endotoxin removal were then completed for the subsequent construction of a stable cell line.

[0382] (1.2) Cell transfection

[0383] (1.2.1) Cell seeding

[0384] HEK-293A cells in the logarithmic phase were digested and diluted to 2×10 5 cell / mL cell suspension, inoculated in 25cm 2 5 mL of cell suspension was inoculated into each plastic culture flask and cultured overnight. Transfection was performed when the cell confluence was approximately 70%.

[0385] (1.2.2) Transient cell transfection

[0386] Transfection was performed when cells reached approximately 70% confluency. Before transfection, remove the culture medium from the flask and add 5 mL of OPTI-MEM medium. Add 3 μg of pcDNA3.1-hLPA plasmid DNA to 500 μL of OPTI-MEM medium and mix thoroughly. Add 20 μL of Lipo2000 to another 500 μL of OPTI-MEM, mix gently, and incubate at room temperature for 5 minutes. Combine the DNA and Lipo2000 suspensions to a total volume of 1 mL, mix gently, and incubate at room temperature for 20 minutes. Add the DNA and Lipo2000 mixture to a 25 cm2 flask, shake the flask back and forth to mix thoroughly, and incubate in an incubator. Four hours after transfection, replace the culture medium with 10 mL of complete medium.

[0387] (1.3) Stable cell line screening

[0388] (1.3.1) Cell screening

[0389] 48 hours after transfection, select cells using medium containing G418 (optimal selection concentration). Replace the selection medium every 2-3 days. When normal cells have completely died, switch to fresh medium without G418. Observe cell growth daily. When cells reach 60% confluence, select again using medium containing G418 (optimal selection concentration).

[0390] (1.3.2) Cell culture and cryopreservation

[0391] When cells reach over 90% confluence, transfer them to culture flasks and continue culturing. Then, screen them every 4-5 days using medium containing G418 (optimal screening concentration). Once a certain number of cells stably expressing the transfected plasmid have been achieved, samples can be collected (approximately 15 days after transfection). After screening, continue culturing at a reduced concentration of G418 in the culture medium. Expand the cells for subsequent screening experiments or freeze them.

[0392] Example 2 Evaluation of in vitro activity of lipoprotein (a) (Lp(a)) siRNA

[0393] In this example, the HEK293-LPA stably transfected cell line prepared in Example 1 was used to evaluate the inhibitory activity of LPA-targeted siRNAs RX001001 to RX001145 on the target gene LPA, with RX000001 used as a negative control.

[0394] Test sample preparation: After centrifugation, dissolve each siRNA sample in DEPC HO according to the specifications of each tube to prepare a 20 μM stock solution. This stock solution is then serially diluted with DEPC HO to produce 0.04 μM and 0.004 μM working solutions. Dose screening experiments are performed at final duplex concentrations of 1 nM and 0.1 nM.

[0395] 96-well transfection and detection: HEK293-LPA stable cell lines grown to near confluence were digested with trypsin, the cells were washed, and a cell suspension was prepared for plating in 96-well plates. 5 μL of 0.04 μM and 0.004 μM working solutions were dispersed in 20 μL Opti-MEM to form an siRNA mixture. 0.2 μL of RNAiMAX was dispersed in 24.8 μL Opti-MEM, incubated for 5 minutes, and then mixed with each siRNA mixture and incubated for 10 minutes to form a transfection complex. 150 μL of cell suspension (12,000 cells) was added to the transfection complex per well, and the 96-well plate was placed in an incubator and cultured for 24 hours.

[0396] Remove the 96-well plate and lyse the cells. Total RNA was extracted using a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction. A reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo (dT) 18 reverse transcription primer was selected. 20 μL of reverse transcription system was prepared according to the method described in the reverse transcription kit instructions and the reverse transcription reaction was completed. Then, the expression level of the target gene mRNA in the cells was detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Roche, LightCycler 480). In this real-time fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene. Primers specific to the target gene and primers specific to the GAPDH internal reference gene were used to detect the target gene and the GAPDH internal reference gene, respectively. The sequences of the detection primers are shown in Table 12.

[0397] Table 12 Sequences of detection primers

[0398] In the real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0399] Table 13 Inhibitory activity of target genes in HEK293-LPA stably transfected cell lines after administration of RX000001-RX001145

[0400] Example 3 Evaluation of the activity of L96 vector-conjugated siRNA in HDI mice

[0401] In this example, the BALB / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the same LPA target siRNA sense strand 3' end conjugated to L96 carrier conjugates RZ001002 to RZ001030 on the target gene LPA, with RZ000002 used as a negative control.

[0402] Plasmid construction:

[0403] Sangon Biotech (Shanghai) Co., Ltd. was commissioned to construct the LPA gene sequence (NM_005577.4) into the pcDNA-CMV vector plasmid to form the pcDNA-CMV-RG001 expression plasmid.

[0404] Mouse model construction:

[0405] The BALB / c mouse hydrodynamic injection model is established by rapidly injecting a pcDNA-CMV-RG001 plasmid solution through the tail vein at high pressure. On day 3 of the experiment, mice were injected with 10 μg of pcDNA-CMV-RG001 via the tail vein hydrodynamic injection over 5 seconds, at an injection volume of 8% of their body weight. The plasmid DNA for injection was diluted with physiological saline, prepared immediately prior to injection, and stored at 4°C.

[0406] Animal grouping, drug administration and tissue sample collection:

[0407] 6-8 week old BALB / c mice (all female) were randomly divided into groups according to body weight, with 5 mice per group. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were dosed according to body weight and administered by subcutaneous injection in the abdomen as a single dose. Each drug conjugate was administered in the form of a 0.1 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 1 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3). On the 4th day (recorded as D4), 5 mice of all groups were sacrificed. The sacrificed mice were subjected to gross dissection and liver tissue of each sacrificed mouse was collected. The liver tissue was cut into small pieces of about 2 mm3 and preserved with RNA later.

[0408] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0409] For each mouse, 1 μg of total RNA was taken respectively, and a reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used and Oligo (dT) 18 reverse transcription primer was selected. According to the method described in the reverse transcription kit instructions, 20 μL reverse transcription system was configured and the reverse transcription reaction was completed. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression level of the target gene mRNA in the animal was then detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as an internal reference gene, and the target gene and the Nero internal reference gene were detected using primers for the target gene and primers for the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 14.

[0410] Table 14 Sequences of detection primers

[0411] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table 11), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0412] Table 15 Inhibitory activity of target genes in mice after administration of siRNA conjugates

[0413] The results of Example 3 showed that at a dose of 1 mg / kg, RZ001003, RZ001004, RZ003005 and RZ001019 had an LPA mRNA inhibitory effect greater than 50%.

[0414] Example 4 Evaluation of Multiple-Dose Activity of L96 Vector-Conjugated siRNA in HDI Mice

[0415] In this example, the BALB / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of the LPA target gene LPA at different doses of the 3' end of the LPA target siRNA sense chain conjugated to the L96 carrier conjugates RZ001002, RZ001003, RZ001004, RZ001005, RZ001006, RZ001008, RZ001009, RZ001012, RZ001018, RZ001019, RZ001026, and RZ001028.

[0416] Animal grouping, drug administration and tissue sample collection:

[0417] 6-8 week old BALB / c mice (all female) were randomly divided into groups according to body weight, with 5 mice per group. The mouse HDI model was established using the same pcDNA-CMV-RG001 plasmid and mouse hydrodynamic injection model construction method as in Example 3. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. All mice were given a single dose of drug conjugates at a subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a PBS solution of 0.05 mg / mL, 0.1 mg / mL, and 0.3 mg / mL (calculated as siRNA), and the administration volume was 10 mL / kg of mouse body weight. That is, the dosage of each drug conjugate was 0.5 mg / kg, 1 mg / kg, and 3 mg / kg of mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without drug conjugate). The day of administration was recorded as day 0 (recorded as D0). Plasmid injection was performed on the 3rd day after administration (recorded as D3). Five mice from all groups were sacrificed on the 4th day (recorded as D4). The mice were killed and their liver tissues were collected. The liver tissues were cut into small pieces of about 2 mm3 and preserved in RNA later.

[0418] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0419] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used to select Oligo (dT) 18 Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription systems and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as an internal reference gene, and the target gene and the Nero internal reference gene were detected using primers for the target gene and primers for the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 10.

[0420] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TMFast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table 11), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0421] The results of Example 3 showed that RZ001002, RZ001003, RZ001004, RZ001005, RZ001006, RZ001008, RZ001009, RZ001012, RZ001018, RZ001019, RZ001026, and RZ001028 could dose-dependently inhibit LPA mRNA expression in mouse liver tissue; at a dose of 3 mg / kg, RZ001004, RZ001005, RZ001006, RZ001008, RZ001009, RZ001012, RZ001018, RZ001026, and RZ001028 reduced LPA mRNA expression by 80% (see Figure 1, Table 16).

[0422] Table 16 Inhibitory activity of target genes in BALB / c-HDI mice after administration of siRNA conjugates

[0423] Example 5 Toxicological evaluation of L96 vector-conjugated siRNA in mice

[0424] In this example, a single subcutaneous injection of 300 mg / kg was used to evaluate the nature and extent of the toxic reactions of the carrier conjugates RZ001003, RZ001004, RZ001005, RZ001018, and RZ001026 in ICR mice.

[0425] Animal grouping, drug administration and tissue sample collection:

[0426] 6-8 week old ICR mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, with 10 mice per group, half male and half female. Each test group was given a predetermined dose of the drug conjugate and a PBS control group was added. The drug dose was calculated based on the body weight of all mice and administered as a single subcutaneous injection in the abdomen. Each drug conjugate was administered as a 30 mg / mL (calculated as siRNA) PBS solution, with a dosing volume of 10 mL / kg mouse body weight, i.e., the dose of each drug conjugate was 300 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (without the drug conjugate) and observed for 14 days. The day of administration was designated as day 0 (denoted as D0). Clinical observations were performed at least once a day after the first dose during the experimental period, and body weight and food intake were measured twice a week. On the 14th day of the experiment (denoted as D14), blood was drawn from all animals (mice were fasted for at least 12 hours before sampling and testing, but water was not allowed) for hematological and blood biochemical tests. Blood was collected and anticoagulated with EDTA-K2, and routine blood tests were performed using an automated modular blood and body fluid analyzer (Sysmex XN-10B1). Blood samples (without anticoagulation) were collected and allowed to stand at room temperature for approximately 30 minutes. After blood coagulation, they were centrifuged at 2000 g for 10 minutes at 4°C, and blood biochemical analysis was performed using a fully automated biochemical analyzer (Sysmex BX-4000). All animals were dissected on day 14 of the experiment (designated D14). Mice were fasted for at least 12 hours prior to dissection and anesthetized with Zotal 50 (Vicco, 8G4VA, France). After blood collection, the animals were euthanized by exsanguination of the abdominal aorta for gross anatomical examination. Histopathological examination of the liver and kidneys of all animals was performed using hematoxylin-eosin staining. The results are shown in Figure 2. None of the test substances had significant effects on the general condition, body weight, and gross anatomical observations of ICR mice. Glandular duct-like tissue formation, fibrous tissue proliferation, and inflammatory cell infiltration were observed in the RZ001005 300 mg / kg group; mild perivascular hepatocyte necrosis and inflammatory cell infiltration were observed in the RZ001026 300 mg / kg group; slight increased mitosis was observed in the livers of some animals in each group; no pathological changes related to the test substances were observed in the kidneys.

[0427] Example 6 Evaluation of the activity of CR01008 vector-conjugated siRNA in HDI mice

[0428] In this example, a BALB / c mouse hydrodynamic injection model was used to evaluate the inhibitory activity of siRNA conjugates such as RZ001031, the 3' end of the siRNA sense chain of the same LPA target site conjugated to the CR01008 vector conjugate, against the target gene LPA. RZ000001 was used as a negative control, and Olpasiran was used as a positive control (the sense chain of Olpasiran is (NAG25)sCmsAmGmCmCmCmCmUmUfAfUfUmGmUmUmAmUmAmCmGms(invdA), with a nucleotide sequence as shown in SEQ ID NO. 303; the antisense chain is UmsCfsGmUfAmUfAmAmCmAmAmUfAmAfGmGfGmGfCmsUfsGm, with a nucleotide sequence as shown in SEQ ID NO. 304).

[0429] Animal grouping, drug administration and tissue sample collection:

[0430] 6-8 week old BALB / c mice were randomly divided into groups (all female) by weight, with 5 mice per group. The mouse HDI model was established using the same pcDNA-CMV-RG001 plasmid and mouse hydrodynamic injection model construction method as in Example 3. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose was calculated based on the body weight of all mice and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.1 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the dosage of each drug conjugate was 1 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PBS solution (not containing drug conjugate). The day of administration was recorded as day 0 (recorded as D0), and plasmid injection was performed on the 3rd day after administration (recorded as D3). Five mice from all groups were sacrificed on the 4th day (recorded as D4). The mice were killed and their liver tissues were collected. The liver tissues were cut into small pieces of about 2 mm3 and preserved in RNA later.

[0431] For each mouse, an appropriate amount of liver tissue sample was taken from RNA later, and the liver tissue sample was disrupted in a Tissuelyser II fully automatic tissue homogenizer for 60 seconds. Then, total RNA was extracted using a fully automatic nucleic acid extractor (purchased from Zhejiang Hanwei Technology Co., Ltd.) and a nucleic acid extraction kit (purchased from Zhejiang Hanwei Technology Co., Ltd., GO-MNTR-100) according to the standard operating procedures for total RNA extraction.

[0432] For each mouse, 1 μg of total RNA was collected and reverse transcription kit (Thermo Fisher Scientific, RevertAid First Strand cDNA Synthesis Kit, K1622) was used to select Oligo (dT) 18 Reverse transcription primers were prepared according to the method described in the reverse transcription kit instructions to configure 20 μL reverse transcription systems and complete the reverse transcription reaction. After the reaction was completed, 60 μL RNase-Free water was added to the reverse transcription system to obtain a cDNA solution. The expression of the target gene mRNA in the animal was then detected using a real-time fluorescence quantitative PCR kit (Thermo Fisher Scientific, TaqMan Fast Advanced Master Mix, 4444557) and a fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384). In this real-time fluorescence quantitative PCR method, the Nero gene on the plasmid backbone was used as an internal reference gene, and the target gene and the Nero internal reference gene were detected using primers for the target gene and primers for the Nero internal reference gene, respectively. The sequences of the detection primers are shown in Table 10.

[0433] According to the instructions of the real-time fluorescence quantitative PCR kit, 10 μL of real-time PCR reaction system was prepared for each PCR detection well. Each reaction system contained 4 μL of the cDNA solution obtained by the reverse transcription reaction, 5 μL of TaqMan TM Fast Advanced Master Mix (2×), 0.15 μL 10 μM upstream primer, 0.15 μL 10 μM downstream primer (primer information see Table 10), 0.15 μL 10 μM probe primer, 0.55 μL RNase-Free H2O. The configured reaction system was placed on a real-time fluorescence quantitative PCR instrument (Bio-Rad, CFX Opus 384), and Real-time PCR amplification was performed using a two-step method. The amplification program was 50°C for 2 min, followed by pre-denaturation at 95°C for 20 s, denaturation at 95°C for 3 s, annealing and extension at 60°C for 30 s, and the denaturation, annealing and extension process was repeated for 40 cycles. In this real-time fluorescence quantitative PCR method, the expression level and inhibition rate of the target gene mRNA in each test group were relatively quantitatively calculated using the ΔΔCt method according to the technical method described in the embodiment.

[0434] Table 17 Inhibitory activity of target genes in BALB / c-HDI mice after administration of siRNA conjugates

[0435] The results of Example 6 showed that at a dose of 1 mg / kg, different modified CR01008 vector conjugate siRNAs could significantly inhibit LPA mRNA expression in animal liver tissue, among which RZ001031, RZ001032, RZ001033, and RZ001034 were better than Olpasiran in reducing mRNA levels (see Figure 3, Table 17).

[0436] Example 7 Construction of hApo(a) transgenic mice

[0437] hApo(a) transgenic mice were developed by Saiye (Suzhou) Biotechnology Co., Ltd. based on its unique Conditional knock-in human Apo(a) transgenic mice constructed using the platform.

[0438] (1) Targeting vector design and construction: The human LPA gene was constructed into a vector to form a CAG promoter-loxP-PGK-Neo-6*SV40pA-loxP-Kozak-Human LPA CDS-rBG pA targeting vector. This vector can insert the human LPA gene (NM_005577.4) into the intergenic region between the Eif4enif1 and Drg1 genes at the Hipp11 locus on mouse chromosome 11 in vivo. The targeting vector was verified by enzyme digestion, PCR, and sequencing. After the targeting vector was verified, plasmid extraction and linearization were performed.

[0439] (2) Electroporation of the targeting vector into embryonic stem cells and screening for positive clones: The targeting vector was electroporated into mouse embryonic stem (ES) cells, and positive selection was performed using Neo and negative selection was performed using DTA. Drug-resistant clones were selected, and ES cells were verified using PCR, karyotype analysis, and Southern blot.

[0440] (3) Mouse production: Positive ES cells are microinjected, and the injected blastocysts are transferred into pseudopregnant female mice. After birth, the mice are identified and tested to obtain TurboKnockout mice with reproductive succession.

[0441] (4) Obtaining hApo(a) transgenic mice: Liver-specific Cre-expressing mice were hybridized with TurboKnockout mice, the resistance gene in the middle of the loxP site was removed, and the expression of the human LPA gene was activated to obtain hApo(a) transgenic mice.

[0442] Example 8 Evaluation of the Protein-Lowering Effect of CR01008 Vector-Conjugated siRNA in hApo(a) Transgenic Mice

[0443] In this example, an enzyme-linked immunosorbent assay (ELISA) was used to determine the expression of Apo(a) protein in the serum of mice at different time points after a single administration of CR01008 vector conjugates RZ001031, RZ001032, RZ001033, RZ001034, RZ001035, RZ001038, RZ001039, and RZ0030044, using Olpasiran as a control.

[0444] Animal grouping, drug administration and tissue sample collection:

[0445] Apo(a) transgenic mice (constructed by Saiye (Suzhou) Biotechnology Co., Ltd. on behalf of Suzhou Xuanjing Biotechnology Co., Ltd.) were grouped according to the level of Apo(a) in serum. Each test group was given a predetermined dose of drug conjugate and a PBS control group was added. The drug dose for all mice was calculated based on their body weight and a single dose was administered by subcutaneous injection in the abdomen. Each drug conjugate was administered in the form of a 0.1 mg / mL (calculated as siRNA) PBS solution, and the administration volume was 10 mL / kg mouse body weight, that is, the administration dose of each drug conjugate was 1 mg / kg mouse body weight (calculated as siRNA). The PBS control group was given the same volume of PB11), and the sera of mice in all groups were collected on the 28th day (denoted as D28), the 35th day (denoted as D35), the 42nd day (denoted as D42), the 49th day (denoted as D49), the 56th day (denoted as D56), the 63rd day (denoted as D63), and the 70th day (denoted as D70), and the Apo(a) protein expression was determined using a human Lipoprotein A ELISA kit (Abcam, ab212165).

[0446] Table 18 Changes in human Apo(a) protein levels in transgenic mice after administration of siRNA

[0447] The results of Example 8 showed that, at a single dose of 1 mg / kg, RZ001031, RZ001032, RZ001033, RZ001034, RZ001035, RZ001038, and RZ001039 were all able to significantly reduce the Apo(a) protein level in Apo(a) transgenic mice; it was observed that on day 28, RZ001032 reduced the Apo(a) protein level by 90%, while Olpasiran had an Apo(a) protein reduction effect of 75%; it was observed that on day 56, RZ001032 reduced the Apo(a) protein level by 50%, while Olpasiran had an Apo(a) protein reduction effect of 30%; it was observed that on day 70, the Apo(a) protein level in the RZ001032 group had not yet returned to the pre-drug value (see Figure 4, Table 18).

[0448] Example 9 Evaluation of the protein-lowering effect of CR01008 vector-conjugated siRNA in normal cynomolgus monkeys

[0449] 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.

[0450] Animal grouping, drug administration and tissue sample collection:

[0451] Healthy cynomolgus monkeys aged 2.5-8 years were grouped 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 based on body weight, and a single dose was administered by subcutaneous injection of 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 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.

[0452] Table 19 Changes in LPA protein levels in cynomolgus monkey serum after administration of siRNA conjugates

[0453] The results of Example 9 showed that, at a single 3 mg / kg dose, R301078, R301079, R301081, R301082, R301083, R301084, and R301085 significantly reduced serum LPA protein levels in cynomolgus monkeys. R301078 achieved a maximum inhibitory effect of 97.20% on serum LPA protein (D29), with the protein-lowering effect maintained above 90% from D22 to D57 after administration. Protein levels began to rise again on D64 after administration, and the inhibitory effect remained at 78.26% on D92 (see Figure 5 and Table 19).

[0454] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples and the features of different embodiments or examples described in this specification without contradiction.

[0455] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure. Industrial Applicability

[0456] The double-stranded oligonucleotides and pharmaceutical compositions provided herein have demonstrated excellent LPA gene expression regulation activity in in vitro cell experiments and in mouse models. The double-stranded oligonucleotides disclosed herein have also demonstrated excellent LPA mRNA inhibition activity in LPA transgenic cell lines. The double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates provided herein are capable of effectively regulating the expression level of the target gene LPA, and thus are effective in treating and / or preventing disease symptoms associated with the level of mRNA expressed by the LPA gene, demonstrating promising application prospects.

Claims

1. A double-stranded oligonucleotide for inhibiting LPA gene expression, characterized in that: The double-stranded oligonucleotide comprises a sense strand and an antisense strand; Wherein, the positive strand comprises a nucleotide sequence that is identical or substantially identical to at least 15 consecutive nucleotides in the sequence shown in SEQ ID NO.1, wherein the substantially identical means that the nucleotide sequence has no more than 3 nucleotide differences from the at least 15 consecutive nucleotides; Wherein, the antisense strand comprises at least 17 nucleotides and is complementary or substantially complementary to the sense strand to form a duplex region, wherein the substantially complementary strand means that the sense strand and the antisense strand have a mismatch of no more than 3 nucleotides in the duplex region; Each nucleotide of the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides; Optionally, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides, and at least one 2'-O-(CH2) n -O-R1 modified nucleotides, wherein n is selected from 1 or 2, and R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; If R1 contains a substituent, the substitution in R1 means that one or more hydrogen atoms on the C1-C6 alkoxy group or the C1-C6 alkoxy group are replaced by a substituent, and the substituent in R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides or 2'-O-ethoxymethyl modified nucleotides; Optionally, the 2'-O-(CH2) n The -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotides.

2. The double-stranded oligonucleotide according to claim 1, characterized in that The antisense strand is complementary or substantially complementary to 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides in the nucleotide sequence of SEQ ID NO.1, and the substantially complementary refers to that the mismatch in the complementary region does not exceed 3 nucleotides.

3. The double-stranded oligonucleotide according to claim 1, characterized in that The sense strand comprises 15, 16, 17, 18, 19 or 20 consecutive nucleotides in the sequence of SEQ ID NO.1 or a nucleotide sequence having no more than 2 nucleotide differences from the consecutive nucleotides; Optionally, the duplex region is 15-25 nucleotide pairs in length.

4. A double-stranded oligonucleotide for inhibiting LPA gene expression, characterized in that: The double-stranded oligonucleotide comprises a sense strand and an antisense strand; In the direction from the 5' end to the 3' end, nucleotides 2 to 19 of the antisense strand comprise a complementary region to an LPA RNA transcript, wherein the complementary region comprises a sequence of at least 15, at least 16, at least 17 or at least 18 consecutive nucleotides in any one of SEQ ID NOs. 147-291, or a nucleotide sequence that differs from the consecutive nucleotides by 1, 2 or 3 nucleotides; The sense strand comprises a nucleotide sequence that is at least partially complementary or substantially complementary to the antisense strand to form a duplex region; the substantially complementary sequence means that the mismatch between the sense strand and the antisense strand in the duplex region does not exceed 3 nucleotides.

5. The double-stranded oligonucleotide according to claim 4, characterized in that The nucleotide sequence of the antisense strand comprises 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. 147-291, or a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides; Optionally, the antisense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 147-291; Optionally, the antisense strand comprises a nucleotide sequence having 0, 1 or 2 nucleotide differences from any one of SEQ ID NOs. 147-291; Optionally, the antisense strand comprises a nucleotide sequence having 0 or 1 nucleotide difference from any one of SEQ ID NOs. 147-291; Optionally, the antisense strand is selected from or comprises any one of the nucleotide sequences in SEQ ID NO.147-291.

6. The double-stranded oligonucleotide according to claim 4, characterized in that The nucleotide sequence of the sense strand comprises 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. 2-146, or differs from the consecutive nucleotides by 1, 2 or 3 nucleotides; Optionally, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 2-146; Optionally, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0, 1 or 2 nucleotide differences from any one of SEQ ID NOs. 2-146; Optionally, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0 or 1 nucleotide difference from any one of SEQ ID NOs. 2-146; Optionally, the nucleotide sequence of the sense strand is selected from or comprises any one of SEQ ID NO. 2-146.

7. The double-stranded oligonucleotide according to claim 4, characterized in that The double-stranded oligonucleotide is selected from the modified or unmodified double-stranded oligonucleotide shown in any number from RN001001 to RN001145 in Table 1; Optionally, the double-stranded oligonucleotide is selected from one or more of the modified or unmodified double-stranded oligonucleotides shown in the following numbers: RN001008, RN001010, RN001011, RN001025, RN001042, RN001074 and RN001121.

8. The double-stranded oligonucleotide according to claim 4, characterized in that Each nucleotide in the double-stranded oligonucleotide is independently selected from unmodified or modified nucleotides; Optionally, substantially all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides; Optionally, all nucleotides in the double-stranded oligonucleotide are selected from modified nucleotides; Optionally, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: 2'-halogenated modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-optionally substituted C1-C6 alkyl modified nucleotides, 2'-O-(CH2) n -O-R1 modified nucleotides, 2'-O-Si(R2)3 modified nucleotides, 2'-amino modified nucleotides, abasic nucleotides, or quasi-nucleotides; the quasi-nucleotides are selected from one or more of PNA, MNA, BNA, LNA, GNA, TNA, and UNA; If the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide contains a substituent, the substitution in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide means that one or more hydrogen atoms on the C1-C6 alkyl are replaced by a substituent, and the substituent in the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Wherein, n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; if R1 contains a substituent, the substitution in R1 means that one or more hydrogen atoms on the C1-C6 alkyl or C1-C6 alkoxy are replaced by a substituent, and the substituent in R1 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Each R2 is independently selected from an optionally substituted C1-C6 alkyl group; if R2 contains a substituent, the substitution in R2 means that one or more hydrogen atoms on the C1-C6 alkyl group are replaced by a substituent, and the substituent in R2 is independently selected from a C1-C3 alkyl group, a C1-C3 alkoxy group or a halogen; optionally, the 2'-halogenated modified nucleotide is selected from a 2'-fluoro-modified nucleotide; Optionally, the 2'-O-optionally substituted C1-C6 alkyl modified nucleotide is selected from 2'-O-methyl modified nucleotides; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotide is selected from 2'-O-methoxyethyl modified nucleotide, 2'-O-ethoxymethyl modified nucleotide or 2'-O-2,2,2-trifluoroethoxymethyl modified nucleotide; Optionally, the 2'-O-(CH2) n -O-R1 modified nucleotides are selected from 2'-O-methoxyethyl modified nucleotides; Optionally, the 2'-O-Si(R2)3 modified nucleotide is selected from a 2'-O-TBDMS modified nucleotide, a 2'-O-TIPS modified nucleotide or a 2'-O-TOM modified nucleotide; Optionally, the nucleotides in the double-stranded oligonucleotide are each independently selected from the following modified nucleotides: 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 2'-O-methyl modified nucleotides or 2'-O-methoxyethyl modified nucleotides.

9. The double-stranded oligonucleotide according to claim 8, characterized in that The sense strand and / or the antisense strand contains at least one 2'-O-(CH2) n -O-R1 modified nucleotide or at least one 2'-O-Si(R2)3 modified nucleotide; Optionally, the sense strand and / or the antisense strand contains at least one 2'-O-(CH2) n -O-R1 modified nucleotides.

10. The double-stranded oligonucleotide according to claim 4, characterized in that The sense strand or the antisense strand comprises a 3' overhang having at least 1 nucleotide; Optionally, the antisense strand comprises a 3' overhang of at least 1 nucleotide; Optionally, the sense strand or the antisense strand comprises a 3' overhang of at least 2 nucleotides; Optionally, the antisense strand comprises a 3' overhang of at least 2 nucleotides; Optionally, the sense strand is 18-21 nt in length, and the antisense strand is 19-23 nt in length; Optionally, the sense strand is 19 nt in length and the antisense strand is 21 nt in length; Optionally, the sense strand and / or the antisense strand independently comprises one or more phosphorothioate bonds; Optionally, the sense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 5' end; Optionally, the antisense strand comprises two consecutive phosphorothioate bonds between the terminal nucleotides at the 3' end and the 5' end, respectively.

11. The double-stranded oligonucleotide according to any one of claims 4 to 10, characterized in that The structure of the duplex region is shown in formula (101): SS: 5'-(N)a'-(X)i'-(N)b'-(X)t'-(N)c'-(X)r'-(N)d'-3' AS: 3'-(N)a-(X)i-(N)b-(X)t-(N)c-5' Formula (101) Wherein, SS represents the sense strand, and AS represents the antisense strand; Each N is independently selected from a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide or a 2'-deoxy modified nucleotide; Each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotide or 2'-O-Si(R2)3 modified nucleotide; wherein n is selected from 1 or 2, R1 is selected from optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkoxy; each R2 is independently selected from optionally substituted C1-C6 alkyl; a, a', i, i', b, b', t, t', c, c', r', d' independently represent the number of nucleotides; wherein a' is selected from an integer of 3-8, i' is selected from an integer of 0-3, b' is selected from an integer of 4-13, t' is selected from an integer of 0-4, c' is selected from an integer of 3-9, r' is selected from an integer of 0-3, d' is selected from an integer of 0-9, a is selected from an integer of 4-7, i is selected from an integer of 0-1 b is an integer selected from 4 to 8, t is an integer selected from 0 to 4, and c is an integer selected from 6 to 10; and i', t', r', i, and t are not 0 at the same time, and 0≤t'+r'≤4; Optionally, a' is selected from an integer of 3-8, i' is selected from 0 or 1, b' is selected from an integer of 4-13, t' is selected from 0 or 1, c' is selected from an integer of 3-9; r' is selected from 0 or 1, d' is selected from an integer of 1-8, a is selected from an integer of 4-7, i is selected from 1, b is selected from an integer of 4-8, t is selected from 0 or 1, and c is selected from an integer of 6-10; Optionally, each X is independently selected from 2'-O-(CH2) n -O-R1 modified nucleotides; Optionally, each X is independently selected from a 2'-O-methoxyethyl modified nucleotide or a 2'-O-ethoxymethyl modified nucleotide; Optionally, the duplex region comprises at least one 2'-O-methoxyethyl modified nucleotide or at least one 2'-O-ethoxymethyl modified nucleotide; Optionally, the sense strand is 18-21 nucleotides long, and the antisense strand is 19-23 nucleotides long; Optionally, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides of the nucleotides at positions 7 to 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 or 2'-O-methoxyethyl-modified nucleotides; 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides or 2'-O-methoxyethyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides of the nucleotides at positions 7-10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro-modified nucleotides, at most two nucleotides of the nucleotides at positions 5, 12, and 18 are selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; 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 9-12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, at least three nucleotides of 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; 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 9-12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, in the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; 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 to 12 is selected from 2'-fluoro-modified nucleotides, the nucleotide at position 15 is selected from 2'-O-methoxyethyl-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; Optionally, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, wherein the modifications of the sense strand and the antisense strand are selected from any one of the following (1)-(10): (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 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; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 9 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; the nucleotides at positions 2, 6, 11, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (3) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from 2'-fluoro-modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (4) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (5) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 10, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (6) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 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; the nucleotides at positions 2, 6, 11, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (7) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 in the nucleotide sequence of the sense strand are selected from 2'-fluoro-modified nucleotides, and the remaining positions are selected from The nucleotides at positions 2, 6, 12, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl-modified nucleotides; (8) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 9 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; (9) In the direction from the 5' end to the 3' end, the nucleotide at position 5 of the nucleotide sequence in the sense strand is selected from a 2'-O-methoxyethyl modified nucleotide, the nucleotides at positions 7 to 10 are selected from a 2'-fluoro modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; the nucleotides at positions 2, 6, 11, 14, and 16 of the nucleotide sequence in the antisense strand are selected from a 2'-fluoro modified nucleotide, the nucleotide at position 15 is selected from a 2'-O-methoxyethyl modified nucleotide, and the nucleotides at the remaining positions are selected from a 2'-O-methyl modified nucleotide; (10) In the direction from the 5' end to the 3' end, the nucleotides at positions 7 to 10 of the nucleotide sequence in the sense strand are selected from 2'-fluoro modified nucleotides, the nucleotides at positions 12 and 18 are selected from 2'-O-methoxyethyl modified nucleotides, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; the nucleotides at positions 2, 6, 11, 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, and the nucleotides at the remaining positions are selected from 2'-O-methyl modified nucleotides; Optionally, in the direction from 5' end to 3' end, the terminal nucleotides at the 5' end of the sense strand contain 1 or 2 consecutive phosphorothioate bonds; and / or, the 5' end and 3' end of the antisense strand each independently contain 1 or 2 consecutive phosphorothioate bonds.

12. The double-stranded oligonucleotide according to claim 11, characterized in that Each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and the sense strand is selected from any one of the following A1-A11; and / or, the antisense strand is selected from any one of the following B1-B41: The positive strand: from the 5' end to the 3' end, A1: GmsGmsCmUmUmGmAfUfCfAfUmGmAmAmCmUmAmCmUm; A2: GmsCmsAmGmCmUmCfCfUfUfAmUmUmGmUmUmAmUmAm; A3: GmsCmsUmCmCmUmUfAfUfUfGmUmUmAmUmAmCmGmAm; A4: CmsGmsGmUmAmAmUfGfGfAfCmAmGmAmGmUmUmAmUm; A5: CmsUmsGmGmCmUmUfGfAfUfCmAmAmGmAmAmCmUmAm; A6: AmsGmsAmGmGmAmCfAfAfCmAmGmAmAmUmAmUmUmAm; A7: AmsGmsAmGmG(moe)AmCfAfAfCmAmGmAmAmUmAmUmUmAm; A8: AmsGmsAmGmGmAmCfAfAfCfAmGmAmAmUmAmUmUmAm; A9: CmsUmsGmAmCmAmCfAfAfUfGmCmUmCmAmGmAmAmAm; A10: CmsUmsGmAmC(moe)AmCfAfAfUfGmCmUmCmAmGmAmAmAm; A11: CmsUmsGmAmCmAmCfAfAfUfGmC(moe)UmCmAmGmAmA(moe)Am; The antisense strand: in the direction from the 5'-end to the 3'-end, B1: AmsGfsUmAmGmUfUmCmAmUmGfAmUmCfAmAfGmCmCmsAmsGm; B2: AmsGfsUmAmGmUfUmCmAmUmGfAmUmCfA(moe)AfGmCmCmsAmsGm; B3: UmsAfsUmAmAmCfAmAmUmAmAfGmGmAfGmCfUmGmCmsCmsAm; B4: UmsAfsUmAmAmCfAmAmUmAmAfGmGmAfG(moe)CfUmGmCmsCmsAm; B5: UmsCfsGmUmAmUfAmAmCmAmAfUmAmAfGmGfAmGmCmsUmsGm; B6: AmsUfsAmAmCmUfCmUmGmUmCfCmAmUfUmAfCmCmGmsUmsGm; B7: AmsUfsAmAmCmUfCmUmGmUmCfCmAmUfT(moe)AfCmCmGmsUmsGm; B8: UmsAfsGmUmUmCfUmUmGmAmUfCmAmAfGmCfCmAmGmsCmsAm; B9: UmsAfsGmUmUmCfUmUmGmAmUfCmAmAfG(moe)CfCmAmGmsCmsAm; B10: UmsAfsAmUmAmUfUmCmUmGmUfUmGmUfCmCfUmCmUmsGmsAm; B11: UmsAfsAmUmAmUfUmCmUmGmUfUmGmUfC(moe)CfUmCmUmsGmsAm; B12: UmsUfsUmCmUmGfAmGmCmAmUfUmGmUfGmUfCmAmGmsAmsUm; B13: UmsUfsUmCmUmGfAmGmCmAmUfUmGmUfG(moe)UfCmAmGmsAmsUm; B14: AmsGfsUmAmGmUfUmCmAfUmGmAmUmCfAmAfGmCmCmsAmsGm; B15: AmsGfsUmAmGmUfUmCmAmUmGmAfUmCfAmAfGmCmCmsAmsGm; B16: AmsGfsUmAmGmUfUmCmAfUmGmAmUmCfA(moe)AfGmCmCmsAmsGm; B17:AmsGfsUmAmGmUfUmCmAmUmGmAfUmCfA(moe)AfGmCmCmsAmsGm; B18:UmsAfsUmAmAmCfAmAmUfAmAmGmGmAfGmCfUmGmCmsCmsAm; B19:UmsAfsUmAmAmCfAmAmUmAmAmGfGmAfGmCfUmGmCmsCmsAm; B20:UmsAfsUmAmAmCfAmAmUfAmAmGmGmAfG(moe)CfUmGmCmsCmsAm; B21:UmsAfsUmAmAmCfAmAmUmAmAmGfGmAfG(moe)CfUmGmCmsCmsAm; B22:UmsCfsGmUmAmUfAmAmCfAmAmUmAmAfGmGfAmGmCmsUmsGm; B23:UmsCfsGmUmAmUfAmAmCmAmAmUfAmAfGmGfAmGmCmsUmsGm; B24:UmsCfsGmUmAmUfAmAmCfAmAmUmAmAfG(moe)GfAmGmCmsUmsGm; B25:UmsCfsGmUmAmUfAmAmCmAmAmUfAmAfG(moe)GfAmGmCmsUmsGm; B26:AmsUfsAmAmCmUfCmUmGfUmCmCmAmUfUmAfCmCmGmsUmsGm; B27:AmsUfsAmAmCmUfCmUmGmUmCmCfAmUfUmAfCmCmGmsUmsGm; B28:AmsUfsAmAmCmUfCmUmGfUmCmCmAmUfU(moe)AfCmCmGmsUmsGm; B29:AmsUfsAmAmCmUfCmUmGmUmCmCfAmUfU(moe)AfCmCmGmsUmsGm; B30:UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfGmCfCmAmGmsCmsAm; B31:UmsAfsGmUmUmCfUmUmGmAmUmCfAmAfGmCfCmAmGmsCmsAm; B32:UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfG(moe)CfCmAmGmsCmsAm; B33:UmsAfsGmUmUmCfUmUmGmAmUmCfAmAfG(moe)CfCmAmGmsCmsAm; B34:UmsAfsAmUmAmUfUmCmUfGmUmUmGmUfCmCfUmCmUmsGmsAm; B35: UmsAfsAmUmAmUfUmCmUmGmUmUfGmUfCmCfUmCmUmsGmsAm; B36: UmsAfsAmUmAmUfUmCmUfGmUmUmGmUfC(moe)CfUmCmUmsGmsAm; B37: UmsAfsAmUmAmUfUmCmUmGmUmUfGmUfC(moe)CfUmCmUmsGmsAm; B38: UmsUfsUmCmUmGfAmGmCfAmUmUmGmUfGmUfCmAmGmsAmsUm; B39: UmsUfsUmCmUmGfAmGmCmAmUmUfGmUfGmUfCmAmGmsAmsUm; B40: UmsUfsUmCmUmGfAmGmCfAmUmUmGmUfG(moe)UfCmAmGmsAmsUm; B41:UmsUfsUmCmUmGfAmGmCmAmUmUfGmUfG(moe)UfCmAmGmsAmsUm; 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; the combination mark (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; Optionally, the double-stranded oligonucleotide is selected from any one of the following combinations of the sense strand and the antisense strand: RX001149-RX001192: RX001149: A1 and B1; RX001150: A1 and B2; RX001151: A2 and B3; RX001152: A2 and B4; RX001153: A3 and B5; RX001154: A4 and B6; RX001155: A4 and B7; RX001156: A5 and B8; RX001157: A5 and B9; RX001158: A6 and B10; RX001159: A6 and B11; RX001160: A7 and B11; RX001161: A9 and B12; RX001162: A9 and B13; RX001163: A10 and B13; RX001164: A11 and B13; RX001165: A1 and B14; RX001166: A1 and B15; RX001167: A1 and B16; RX001168: A1 and B17; RX001169: A2 and B18; RX001170: A2 and B19; RX001171: A2 and B20; RX001172: A2 and B21; RX001173: A3 and B22; RX001174: A3 and B23; RX001175: A3 and B24; RX001176: A3 and B25; RX001177: A4 and B26; RX001178: A4 and B27; RX001179: A4 and B28; RX001180: A4 and B29; RX001181: A5 and B30; RX001182: A5 and B31; RX001183: A5 and B32; RX001184: A5 and B33; RX001185: A8 and B34; RX001186: A8 and B35; RX001187: A8 and B36; RX001188: A8 and B37; RX001189: A9 and B38; RX001190: A9 and B39; RX001191: A9 and B40; RX001192: A9 and B41.

13. The double-stranded oligonucleotide according to claim 4, characterized in that The double-stranded oligonucleotide comprises a sense strand and an antisense strand; in the direction from the 5' end to the 3' end, nucleotides 2 to 19 of the antisense strand comprise a complementary region to an LPA RNA transcript, wherein the complementary region comprises a sequence of at least 15, at least 16, at least 17 or at least 18 consecutive nucleotides in any sequence of SEQ ID NO. 167, 188, 196, 202, 215, 220, 245 or 285, or a nucleotide sequence having 1, 2 or 3 nucleotide differences from the consecutive nucleotides; Optionally, the antisense strand comprises a nucleotide sequence having 0, 1, 2 or 3 nucleotide differences from any one of SEQ ID NOs. 167, 188, 196, 202, 215, 220, 245 or 285; and / or, the nucleotide sequence of the sense strand comprises a nucleotide sequence having 0, 1 or 2 nucleotide differences from any one of SEQ ID NO. 22, 43, 51, 57, 70, 75, 100 or 140; Optionally, each nucleotide of the double-stranded oligonucleotide is independently selected from modified nucleotides, and the sense strand is selected from any one of the following A5, A8 or A12-A17; and / or, the antisense strand is selected from any one of the following B42-B49: The positive strand: from the 5' end to the 3' end, A5: CmsUmsGmGmCmUmUfGfAfUfCmAmAmGmAmAmCmUmAm A8: AmsGmsAmGmGmAmCfAfAfCfAmGmAmAmUmAmUmUmAm A12: CmsCmsUmAmGmAmGfGfCfUfCmCmUmUmCmCmGmAmAm A13: CmsAmsGmGmAmCmUfGfCfUfAmCmUmAmCmCmAmUmUm A14: GmsAmsAmUmCmCmAfGfAfUfGmCmUmGmAmGmAmUmUm A15: GmsGmsAmAmGmGmAfCfAfUfGmUmCmAmGmUmCmUmUm A16:GmsGmsAmAmGmAmAfcfAfUfGmUmCmAmGmUmCmUmUm A17: GmsGmsAmAmGmGmAfCfAfUfGmUmCmAmAmUmCmUmUm; The antisense strand: from the 5' end to the 3' end, B42:UmsAfsGmUmUmCfUmUmGfAmUmCmAmAfGmCfCmAmGms(NM054)s(NM054) B43:UmsUfsCmGmGmAfAmGmGfAmGmCmCmUfC(moe)UfAmGmGms(NM054)s(NM054) B44:AmsAfsUmGmGmUfAmGmUfAmGmCmAmGfU(moe)CfCmUmGms(NM054)s(NM054) B45:AmsAfsUmCmUmCfAmGmCfAmUmCmUmGfG(moe)AfUmUmCms(NM054)s(NM054) B46:AmsAfsGmAmCmUfGmAmCfAmUmGmUmCfC(moe)UfUmCmCms(NM054)s(NM054) B47:UmsAfsAmUmAmUfUmCmUfGmUmUmGmUfC(moe)CfUmCmUms(NM054)s(NM054) B48:AmsAfsGmAmCmUfGmAmCfAmUmGmUmUfC(moe)UfUmCmCms(NM054)s(NM054) B49:AmsAfsGmAmUmUfGmAmCfAmUmGmUmCfC(moe)UfUmCmCms(NM054)s(NM054) 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; the combination mark (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; (NM054) represents a nucleotide with the structural formula Optionally, the double-stranded oligonucleotide is selected from any one of the following combinations of the sense strand and the antisense strand: R201078: A5 and B42; R201079: A12 and B43; R201080: A13 and B44; R201081: A14 and B45; R201082: A15 and B46; R201083: A8 and B47; R201084: A16 and B48; R201085:A17 and B49.

14. The double-stranded oligonucleotide according to claim 4, characterized in that The double oligonucleotide is siRNA.

15. A conjugate, characterized in that The conjugate comprises a double-stranded oligonucleotide according to any one of claims 1 to 14 and one or more ligands capable of binding to a cell surface receptor; Optionally, the 3' end and / or 5' end of the sense strand and / or the antisense strand is conjugated to the ligand; Optionally, the 3' end and / or 5' end of the sense strand is conjugated to the ligand; Optionally, the 3' end of the sense strand is conjugated to the ligand; Optionally, the ligand is linked to the 3' end and / or 5' end of the sense strand and / or antisense strand via a phosphate group or a phosphorothioate group; Optionally, the number of the ligands is selected from 1, 2, 3 or 4; Optionally, the number of the ligand is 1; Optionally, the number of the ligand is 1, and the ligand is conjugated to the 3' end of the sense strand; Optionally, the ligand specifically targets liver tissue; Optionally, the ligand is an asialoglycoprotein receptor ligand; Optionally, the ligand comprises galactose or a derivative thereof; Optionally, the derivative of galactose is N-acetylgalactosamine.

16. The conjugate according to claim 15, characterized in that The structure of the conjugate is shown in formula (201): Wherein, Nu represents the double oligonucleotide; Each R ligand independently selected from ASGPR ligands; y is selected from 1, 2, 3 or 4; each of said R ligand are independently conjugated to the 3' end and / or 5' end of the sense strand and / or the antisense strand, and each of the R ligand The conjugation positions are different; Optionally, y is selected from 1, 2, 3 or 4; Optionally, y is selected from 1 or 2; Optionally, y is 1; Optionally, y is 1, and R ligand conjugated to the 3' end of the sense strand; Optionally, y is 2; Optionally, y is 2, and the two R ligand conjugated to the 3' and 5' ends of the sense strand, respectively.

17. The conjugate according to claim 16, characterized in that Each R ligand Each is independently selected from a molecule having a structure as shown in formula (301) or a pharmaceutically acceptable salt or a stereoisomer thereof: Wherein, * represents the conjugation site of the ligand on the sense strand or the antisense strand; j is selected from 1, 2, 3 or 4; Each Z is independently selected from hydroxyl or thiol; Each p is independently selected from 1, 2 or 3; Each q is independently selected from 1, 2 or 3; Each R3 is independently selected from H, an optionally substituted C1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group; if R3 contains a substituent, the substitution in R3 means that one or more hydrogen atoms on the C1-C6 alkyl group or the C1-C6 alkoxy group are replaced by a substituent, and the substituent in R3 is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Each L is independently selected from optionally substituted C1-C 20 Alkylene or R La and R Lb are independently selected from optionally substituted C 1-10 Alkylene, k is selected from 1, 2, 3, 4 or 5; if L contains a substituent, the substitution in L refers to C1-C 20 Alkylene or C 1-10 One or more hydrogen atoms on the alkylene group are substituted by a substituent, and the substituent in L is independently selected from C1-C3 alkyl, C1-C3 alkoxy or halogen; Each Y is independently selected from O, S or NH; Optionally, j is selected from 1, 2 or 3; Optionally, j is 3; Optionally, Z is hydroxy; Optionally, p is 1; Optionally, q is 1; Optionally, each R3 is H; Optionally, each L is independently selected from C1-C 10 Alkylene or Among them, R La and R Lb Independently selected from C1-C5 alkylene, k is 1, 2 or 3; Optionally, k is 1; Optionally, each L is independently selected from Optionally, Y is O; Optionally, each R ligand Independently selected from molecules having a structure as shown in formula (302) or pharmaceutically acceptable salts or stereoisomers thereof: Optionally, each R ligand Independently selected from molecules having the structure shown below or pharmaceutically acceptable salts or stereoisomers thereof: Optionally, each R ligand Independently selected from molecules having any of the following structures or pharmaceutically acceptable salts or stereoisomers thereof:

18. The conjugate according to claim 16, characterized in that The Nu is selected from any one of RX001149-RX001192; Optionally, Nu is selected from any one of RX001149-RX001192, the ligand is selected from the formula (CR01008×3), y=1, and the ligand is conjugated to the 3′ end of the sense strand of the double-stranded oligonucleotide.

19. The conjugate according to claim 16, characterized in that The Nu is selected from any one of R201078-R201085; Optionally, Nu is selected from any one of R201078-R201085, the ligand is selected from the formula (CR01008×3), y=1, and the ligand is conjugated to the 3′ end of the sense strand of the double-stranded oligonucleotide.

20. Use of the double-stranded oligonucleotide according to any one of claims 1 to 14 or the conjugate according to any one of claims 15 to 19 in the preparation of a medicament for alleviating, preventing and / or treating a disease or condition mediated by the LPA gene; Optionally, the LPA gene-mediated disease or condition comprises a disease associated with the level of mRNA expressed by the LPA gene; Optionally, the disease or condition mediated by the LPA gene comprises cardiovascular disease; Optionally, the cardiovascular disease comprises: Hyperlipoprotein(a)emia, Buerger'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, hyperapolipoproteinβ-lipoproteinemia, 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) containing particles.

21. A pharmaceutical composition, characterized in that Comprising the double-stranded oligonucleotide according to any one of claims 1-14 and / or the conjugate according to any one of claims 15-19.

22. A kit, characterized in that: The kit comprises the double-stranded oligonucleotide according to any one of claims 1 to 14, and / or the conjugate according to any one of claims 15 to 19, and / or the pharmaceutical composition according to claim 21.

23. A method for inhibiting LPA gene expression, characterized in that: The method comprises administering to a subject an effective amount of a double-stranded oligonucleotide according to any one of claims 1 to 14, and / or a conjugate according to any one of claims 15 to 19, and / or a pharmaceutical composition according to claim 21; Optionally, the method comprises contacting a subject's cells with the double-stranded oligonucleotide, and / or the conjugate, and / or the pharmaceutical composition, Optionally, the inhibition of LPA gene expression is inhibition of LPA gene expression by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70%.

24. A method for alleviating, treating and / or preventing a disease or condition mediated by LPA gene, characterized in that: It comprises administering to a subject an effective amount of a double-stranded oligonucleotide according to any one of claims 1 to 14, and / or a conjugate according to any one of claims 15 to 19, and / or a pharmaceutical composition according to claim 21; Optionally, the LPA gene-mediated disease or condition comprises a disease associated with the level of mRNA expressed by the LPA gene; Optionally, the LPA gene-mediated disease or condition is a cardiovascular disease; Optionally, the cardiovascular disease includes: hyperlipoprotein(a)emia, Buerger'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) containing particles.

Images