SiRNA for targeted regulation of LPA gene expression and application thereof

Abstract

The invention provides siRNA for targeted regulation and control of LPA gene expression and application thereof. Cell experiment results show that the oligonucleotide duplex can significantly inhibit the expression of the LPA gene, and can be used for preparing drugs for preventing or treating atherosclerosis and other related diseases.

Description

Technical Field

[0001] The present disclosure belongs to the field of biomedicine, and specifically relates to siRNA targeting and regulating LPA gene expression and its use. Background Art

[0002] The LPA gene encodes apolipoprotein A (Apo(a)) in the human body, a protein that plays a key role in various physiological processes, including lipid metabolism, inflammatory response, and vascular physiological function. In recent years, numerous studies have shown that abnormal expression of the LPA gene is closely associated with the occurrence and development of various diseases.

[0003] In the context of cardiovascular disease, high levels of LPA gene expression are closely associated with the development and progression of atherosclerosis and an increased risk of thrombosis. Apolipoprotein A (Apo(a)) promotes the aggregation and oxidation of lipoprotein particles, leading to impaired endothelial cell function, inflammatory cell infiltration, and the formation and instability of atherosclerotic plaques. Furthermore, LPA gene polymorphisms are associated with susceptibility to cardiovascular diseases such as coronary heart disease and myocardial infarction, further highlighting the potential value of regulating LPA gene expression in the prevention and treatment of cardiovascular diseases.

[0004] Currently, treatment options for regulating LPA gene expression are relatively limited. Traditional drugs for cardiovascular and metabolic diseases primarily work through lipid regulation, anti-inflammatory, and anti-platelet aggregation pathways. However, these drugs often fail to directly target the LPA gene, resulting in limited therapeutic efficacy and potential side effects. Therefore, developing a new method for specifically and efficiently regulating LPA gene expression holds significant clinical significance and market potential.

[0005] Oligonucleotides are a class of short DNA or RNA molecules or oligomers that can bind to their respective complementary oligonucleotides, DNA, or RNA in a sequence-specific manner to form duplexes or, less commonly, higher-order hybrids. Oligonucleotides can sequence-specifically bind to complementary RNA strands. Upon hybridization, they can induce RNase H to cleave the target RNA, thereby reducing target gene expression. Furthermore, in natural oligonucleotides, the nucleotides are linked by phosphodiester bonds. Under physiological conditions, oligonucleotides are particularly sensitive to nucleases. Therefore, when preparing oligonucleotide drugs, natural, unmodified, or unaltered oligonucleotides are prone to rapid degradation in vivo, resulting in very limited activity and poor drugability. Modification of oligonucleotides is an effective way to enhance their activity, increasing their stability to nucleases, their affinity for RNA, and better promoting endocytosis and tissue targeting, thereby effectively regulating target gene expression.

[0006] Currently, oligonucleotides with good inhibitory activity that target and regulate LPA gene expression, especially siRNA, still need to be explored and improved. Summary of the Invention

[0007] To address the existing technical problem of a lack of more effective siRNA duplexes for targeting and regulating LPA gene expression, the present disclosure provides siRNA duplexes for targeting and regulating LPA gene expression and their use in preventing and treating cardiovascular and cerebrovascular diseases. By modifying the base siRNA sequence, the present disclosure screened multiple siRNA modifications that significantly inhibit LPA gene expression and provided corresponding siRNA conjugates.

[0008] The technical solutions disclosed herein include but are not limited to:

[0009] In one aspect, the present disclosure provides a siRNA duplex comprising an oligonucleotide duplex consisting of a pair of sense and antisense strands.

[0010] In another aspect, the present disclosure provides a conjugate for reducing the expression of LPA, comprising the above-mentioned siRNA duplex and a conjugated group connected thereto.

[0011] In another aspect, the present disclosure provides a nucleic acid-protein complex comprising the double-stranded region or the antisense strand of the double-stranded region of the siRNA duplex, and a nuclease.

[0012] In another aspect, the present disclosure provides a recombinant vector comprising a nucleic acid molecule encoding the aforementioned siRNA duplex.

[0013] In some embodiments, the vector backbone of the recombinant vector is selected from a recombinant viroid-derived circular RNA vector, tRNA, rRNA scaffold and chimeric tRNA / pre-mi RNA vector.

[0014] In another aspect, the present disclosure provides a recombinant cell that synthesizes and secretes the aforementioned siRNA duplex.

[0015] In some embodiments, the recombinant cell is selected from Rhodopseudomonas sulfophilus and RNase III-deficient Corynebacterium glutamicum.

[0016] In another aspect, the present disclosure provides a method for preparing siRNA duplexes, comprising culturing the above-mentioned recombinant cell, or chemically synthesizing the siRNA duplexes.

[0017] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned siRNA duplex, the aforementioned conjugate, or the aforementioned nucleic acid-protein complex, and a pharmaceutically acceptable carrier.

[0018] In another aspect, the present disclosure provides a method for inhibiting LPA gene expression, comprising contacting the aforementioned siRNA duplex, the aforementioned conjugate, the aforementioned nucleic acid-protein complex, or the aforementioned pharmaceutical composition with a target cell.

[0019] In some embodiments, the methods are for non-diagnostic or non-therapeutic purposes.

[0020] In some embodiments, the method is in vivo or in vitro.

[0021] In another aspect, the present disclosure provides a use of the aforementioned siRNA duplex, the aforementioned conjugate, the aforementioned nucleic acid-protein complex, or the aforementioned pharmaceutical composition in the preparation of a drug for treating diseases associated with LPA gene expression.

[0022] The LPA gene expression-related disease is selected from the group consisting of Apo(a) protein overexpression, LPA gene pathogenic mutation, Apo(a) protein metabolism abnormality, and diseases caused by abnormal interaction between LPA or Apo(a) and another substance.

[0023] In some embodiments, the LPA gene expression-related disease is selected from Berger's disease, peripheral artery disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic stenosis, aortic regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperlipoprotein betalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and / or any other disease associated with elevated levels of Lp(a) particles and other related conditions, pathologies or syndromes that have not yet been identified.

[0024] In another aspect, the present disclosure provides the siRNA duplex, the conjugate, the nucleic acid-protein complex, or the pharmaceutical composition for use in treating diseases associated with LPA gene expression.

[0025] In another aspect, the present disclosure provides a method for treating a disease associated with LPA gene expression, comprising administering an effective amount of the aforementioned siRNA duplex, the aforementioned conjugate, the aforementioned nucleic acid-protein complex, or the aforementioned pharmaceutical composition to a subject in need thereof.

[0026] In another aspect, the present disclosure provides a use of the aforementioned siRNA duplex, the aforementioned conjugate, the aforementioned nucleic acid-protein complex, or the aforementioned pharmaceutical composition in the preparation of a drug for preventing or treating atherosclerosis and / or cardiovascular and cerebrovascular diseases.

[0027] In another aspect, the present disclosure provides the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid-protein complex, or the above-mentioned pharmaceutical composition for preventing or treating atherosclerosis and / or cardiovascular and cerebrovascular diseases.

[0028] In another aspect, the present disclosure provides a method for preventing or treating atherosclerosis and / or cardiovascular and cerebrovascular diseases, comprising administering an effective amount of the aforementioned siRNA duplex, the aforementioned conjugate, the aforementioned nucleic acid-protein complex, or the aforementioned pharmaceutical composition to a subject in need thereof.

[0029] In another aspect, the present disclosure provides a use of the aforementioned siRNA duplex, the aforementioned conjugate, the aforementioned nucleic acid-protein complex, or the aforementioned pharmaceutical composition in preparing a reagent and a kit for inhibiting LPA gene expression. DETAILED DESCRIPTION

[0030] In order to make this disclosure easier to understand, some terms are first defined. In addition, it should be noted that whenever a value or a range of values for a parameter is listed, it is intended to indicate that values and ranges intermediate to these recited values are also intended to be part of this disclosure.

[0031] As used herein, the articles "a" and "an" refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or more than one element, such as a plurality of elements.

[0032] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to."

[0033] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly dictates otherwise.

[0034] As used herein, the term "approximately" or "approximately" as applied to one or more target values refers to a value similar to the reference value. In certain embodiments, unless otherwise indicated or in addition apparent from context, the term "approximately" or "approximately" refers to a value falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in either direction (greater than or less than) or less (unless such numeral will exceed 100% of possible value).

[0035] As used herein, "LPA" refers to the gene encoding apolipoprotein A or the protein expressed by this gene.

[0036] The term "LPA gene" may refer to a wild-type LPA gene or a mutant LPA gene having sequence variations. Many sequence variations in the LPA gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, ncbi.nlm.nih.gov / snp).

[0037] "G," "C," "A," and "U" each generally represent a nucleotide comprising guanine, cytosine, adenine, and uracil as a base, respectively. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" may also refer to a modified nucleotide (as further described below) or an alternative replacement moiety. The skilled artisan is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide (including a nucleotide having such a replacement moiety). For example, without limitation, a nucleotide comprising inosine as its base can pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide comprising uracil, guanine, or adenine can be replaced in the nucleotide sequence of the present disclosure by a nucleotide comprising, for example, inosine. Sequences comprising such replacement moieties are suitable for use in, but not limited to, double-stranded RNAs, double-stranded RNA modifications, double-stranded RNA conjugates, pharmaceutical compositions, and methods of the present disclosure.

[0038] The terms "complementary," "fully complementary," and "substantially complementary" may be used herein to refer to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA and a target sequence, as will be understood from the context of their use. In some embodiments herein, a first nucleotide sequence is considered complementary to a second nucleotide sequence if the first nucleotide sequence exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to the second nucleotide sequence. In an exemplary embodiment, 18 of the 20 nucleobases of the first nucleotide sequence pair with corresponding regions of the second nucleotide sequence, achieving 90% complementarity.

[0039] The terms "double-stranded RNA," "double-stranded RNA (dsRNA) molecule," "dsRNA," and "RNA duplex" are used interchangeably and specifically refer to a complex of RNA molecules having a double-stranded structure comprising two antiparallel, complementary, or substantially complementary nucleic acid strands, oriented in "sense" and "antisense" directions relative to a target gene, such as the LPA gene. In some embodiments, the double-stranded RNA (dsRNA) triggers the degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi). Herein, in some cases, the term "siRNA duplex" also typically refers to the technical meaning indicated by the above definition.

[0040] As is known in the art, the terms "siRNA duplex," "double-stranded RNAi agent," "RNAi agent," "small interfering RNA," or "siRNA" refer to small interfering RNA RNAi molecules. It is a type of double-stranded RNA molecule, also known in the art as short interfering RNA or silencing RNA. siRNA typically comprises a sense strand (also known as a passenger strand) and an antisense strand (also known as a leader strand), wherein each strand is 17 to 30 nucleotides in length, typically 19 to 25 nucleotides in length, wherein the antisense strand is complementary (such as at least 95% complementary, such as fully complementary) to the target nucleic acid (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand such that the sense strand and the antisense strand form a duplex or duplex region. The sense and antisense strands of the siRNA can form a blunt-ended duplex, or can form a duplex comprising a 3' overhang, which can be, for example, 1, 2, or 3 nucleotides in length, similar to the products produced by Dicer, which can form a RISC substrate in vivo. Effective extended forms of Dicer substrates have been described in US Pat. No. 8,349,809 and US Pat. No. 8,513,207, which are incorporated herein by reference. In some embodiments, both the sense and antisense strands have 3' overhangs of 2 nucleotides in length. Thus, the duplex region can be, for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, such as 19, 20, 21, 22, or 23 nucleotides in length.

[0041] Furthermore, in this article, "siRNA" also refers to "basic sequence" in some cases. In this article, "basic sequence" specifically refers to an siRNA duplex in which each nucleotide in a double-stranded ribonucleic acid is an unmodified nucleotide, and is also referred to as "motif", "siRNA motif" and the like throughout the article. Therefore, in this article, "siRNA", "basic sequence", "motif", "siRNA motif" can be used interchangeably, and their meanings also include the corresponding nucleotide arrangement order of the siRNA duplex referred to. In this article, those skilled in the art can clearly understand the accurate technical meanings they refer to based on the technical meaning of the context. In addition, the 5' terminal nucleotide of the antisense strand of the motif may be connected to a 5' phosphate group or a 5' phosphate derivative group or may not be connected to a 5' phosphate group or a 5' phosphate derivative group.

[0042] In this article, "siRNA modifier" refers to a double-stranded ribonucleic acid comprising at least one modified nucleotide, and in some cases, it also appears as a "double-stranded ribonucleic acid modifier". In this article, the siRNA motif is modified in different modification methods to prepare the corresponding siRNA modifier. For example, in some embodiments, the motif is modified in an alternating modification method to obtain an alternating modified siRNA modifier. In other embodiments, the motif is modified in a modification method using a specific modification template to obtain a siRNA modifier modified with a specific modification template. In yet other embodiments, the motif is modified using the anti-off-target modification method described herein to obtain an anti-off-target modified siRNA modifier. In some cases, a variety of different modification methods can be used simultaneously to modify the same siRNA motif to obtain corresponding siRNA modifiers with a variety of modification methods.

[0043] Herein, "siRNA conjugate" refers to a double-stranded RNA conjugate obtained by linking a conjugating group to a double-stranded RNA or a double-stranded RNA modified substance, or a conjugate of a double-stranded RNA modified substance. Preferably, "siRNA conjugate" refers to a conjugate of a double-stranded RNA modified substance.

[0044] In some cases herein, "siRNA" refers not only to the unmodified siRNA duplex (or siRNA motif) described above, but may also refer to its corresponding siRNA modifications and / or siRNA conjugates. For example, in the context of methods including but not limited to treatment methods, therapeutic agents, etc., siRNA generally refers to at least one of siRNA motifs, siRNA modifications, and / or siRNA conjugates. For those skilled in the art, the specific technical meaning can be clearly understood in conjunction with the context.

[0045] The term "antisense strand" refers to a double-stranded ribonucleic acid (e.g., RNA duplex herein) comprising a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not fully complementary to the target sequence, mispairing can occur within the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is within the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' end and / or 3' end. The target sequence, for example, derives from Homo sapiens lipoprotein (a) (LPA), mRNA, whose accession number is NM_005577.4 in the NCBI database.

[0046] The term "sense strand" as used herein refers to the strand of a double-stranded ribonucleic acid that includes a region that is substantially complementary to a region of the antisense strand (as that term is defined herein).

[0047] The term "nonsense sequence" refers to an artificially designed or selected nucleic acid sequence that does not significantly complementarily bind to the mRNA of any known functional gene in the target organism and, therefore, theoretically cannot induce specific gene silencing. This sequence serves as an experimental control to eliminate the influence of non-target factors (such as transfection stress, vector backbone effects, or innate immune responses) on the results of RNA interference experiments.

[0048] The term "overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded region of a double-stranded RNA when the 3' end of one strand of the double-stranded RNA extends beyond the 5' end of the other strand, or, although less frequently, at least one unpaired nucleotide that protrudes from the double-stranded region of the double-stranded RNA when the 5' end of one strand of the double-stranded RNA extends beyond the 3' end of the other strand. The term "blunt end" means that there are no unpaired nucleotides at either or both ends of the double-stranded RNA, i.e., there are no overhangs. A "blunt-ended" double-stranded RNA refers to a double-stranded region throughout the entire length of the double-stranded RNA, i.e., there are no overhangs at either end of the molecule.

[0049] As used herein, "double-stranded region" refers to a double-stranded region formed by the complementarity or substantial complementarity of the sense strand and the antisense strand of a double-stranded RNA through base pairing, and its length is generally 19, 20, or 21 bases. In some cases, its length may be other lengths other than 19, 20, or 21 bases. Moreover, those skilled in the art will readily understand, based on the conventions in the art, that a length of 19, 20, or 21 bases may also be referred to as a length of 19, 20, or 21 base pairs (bp).

[0050] Therefore, those skilled in the art will understand that for double-stranded RNAs containing overhangs, their structure may include an overhang at the 5' end of the sense strand in the double-stranded region, an overhang at the 3' end of the sense strand in the double-stranded region, or an overhang at both ends of the double-stranded region; for double-stranded RNAs that do not include overhangs, i.e., "blunt-ended" double-stranded RNAs, as described above, the entire length of the double-stranded region is a double-stranded region.

[0051] Those skilled in the art can easily understand that the definitions of "overhanging end", "blunt end", "double-stranded region" and the like described above also apply to double-stranded RNA modifications and double-stranded RNA conjugates.

[0052] The terms "complementary" and "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, namely, the pairing of bases on one strand of a double-stranded RNA molecule with bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T), or in RNA with uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of that strand can be inferred from the sequence of its complementary strand. Correspondingly, a "mismatch" means that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0053] The term "substantially complementary" means that there are no more than 3 base mismatches between the two nucleotide chains involved, that is, there are 1, 2 or 3 base mismatches between the two nucleotide chains involved.

[0054] The terms "complementary" and "substantially complementary" can be used with respect to base pairing between the sense and antisense strands of a double-stranded RNA, or between the antisense strand of a double-stranded RNA and a target gene, as will be understood from the context of their use.

[0055] As used herein, "difference" refers to the presence of nucleotides in the nucleotide sequence of any ribonucleic acid chain that are different from the nucleotide sequence. For example, when describing that the sense chain and / or antisense chain has 1 to 3 nucleotide differences from a specific sequence, it means that the sense chain and / or antisense chain has 1 to 3 different nucleotides from the ribonucleic acid chain shown in the specific sequence.

[0056] The term "alternating modification" or "alternating fluorine-oxygen modification" refers to the use of either 2'-methoxy (2'-OMe) or 2'-fluoro (2'-F) modifications on each nucleotide in the order of the nucleotides in each strand of a double-stranded RNA. For example, in the antisense strand of an siRNA, odd-numbered positions (i.e., positions 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, and 23) are modified with 2'-methoxy, while even-numbered positions (i.e., positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22) are modified with 2'-fluoro. For example, in the sense strand, the nucleotide on the sense strand that pairs with the 2'-methoxy-modified nucleotide on the antisense strand is 2'-fluoro-modified, while the nucleotide on the sense strand that pairs with the 2'-fluoro-modified nucleotide on the antisense strand is 2'-methoxy-modified. In some embodiments, the odd-numbered nucleotides of the sense strand and the even-numbered nucleotides of the antisense strand are both 2'-fluoro-modified nucleotides, and the nucleotides at other positions are 2'-methoxy-modified nucleotides. In some embodiments, the double-stranded RNA modified substance with "alternating modification" or "fluorine-oxygen alternating modification" has a 3',5'-phosphorothioate bond between the first and second nucleotides and between the second and third nucleotides at the 5' end of the sense strand; the first and second nucleotides and between the second and third nucleotides at the 5' end of the antisense strand have a 3',5'-phosphorothioate bond, and the first and second nucleotides and between the second and third nucleotides at the 3' end of the antisense strand have a 3',5'-phosphorothioate bond.

[0057] In RNA interference (RNAi), inhibition of the target gene is achieved by loading the antisense strand of the siRNA onto the AGO2 protein to form a silencing complex (RISC), which then cleaves the gene's transcript, the mRNA. Loading the RISC requires phosphorylation (5'-phosphate) of the 5' end of the antisense strand. 5'-terminal phosphorylation can occur naturally within the cell via cleavage and polyadenylation factor I subunit 1 (Clp1) or through chemical synthesis. The term "natural 5'-terminal phosphorylation" or "simple 5'-terminal phosphorylation" refers to the process by which the 5'-terminal phosphorylation of the siRNA antisense strand occurs within the cell, rather than through chemical synthesis.

[0058] In this article, a "conjugated group" is a GalNAc derivative attached to an oligonucleotide. In some cases, the conjugated group includes a targeting group (also referred to as a ligand), and optionally further includes a linker, such as a GalNAc derivative attached to an oligonucleotide via a linker (e.g., a divalent, trivalent, or tetravalent branched linker), or a GalNAc derivative attached to an oligonucleotide via a monovalent linker. In most cases, "ligand" and "conjugated group" have meanings well known in the art.

[0059] The term "inhibit" as used herein may be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition. In some cases herein, "regulate" refers to "inhibit," and those skilled in the art will clearly understand its specific meaning based on the context.

[0060] As used herein, the phrase "inhibiting the expression of LPA" includes inhibiting the expression of any LPA gene (such as, for example, a mouse LPA gene, a rat LPA gene, a monkey LPA gene, or a human LPA gene), as well as variants (e.g., naturally occurring variants) or mutants of LPA genes. Thus, the LPA gene can be a wild-type LPA gene, a mutant LPA gene, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic LPA gene.

[0061] "Inhibiting LPA gene expression" includes any level of inhibition of the LPA gene, such as at least partial inhibition of LPA gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0062] Based on the level of any variable related to LPA gene expression, such as LPA mRNA level or LPA protein level, LPA gene expression can be assessed. The inhibition can be assessed by a decrease 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 a similar untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0063] In this document, "regulate" can, in some cases, mean the same as "inhibit"; accordingly, "regulate LPA gene expression" can mean "inhibit LPA gene expression." Those skilled in the art will clearly understand the specific technical meaning in the context.

[0064] As used herein, "patient" or "subject" is intended to include humans or non-human animals, preferably mammals, such as monkeys. More preferably, the subject or patient is a human.

[0065] As used herein, "LPA-related disease" is intended to include any disease associated with the LPA gene or protein. Such diseases can be caused, for example, by overproduction of LPA protein, by mutations in the LPA gene, by abnormal cleavage of the LPA protein, by abnormal interactions between LPA and other proteins or other endogenous or exogenous substances. Exemplary LPA-related diseases include 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 associated with elevated levels of Lp(a) particles and other related conditions, pathologies or syndromes not yet identified.

[0066] As used herein, "therapeutically effective amount" is intended to include an amount of a RNAi agent sufficient to achieve treatment of an LPA-associated disease (e.g., by attenuating, ameliorating, or maintaining the existing disease or one or more disease symptoms) when administered to a patient. The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the patient's medical history, age, weight, family history, genetic makeup, stage of the pathological process mediated by LPA expression, type of previous or concomitant therapy (if any), and other individual characteristics of the patient being treated.

[0067] As used herein, a "prophylactically effective amount" refers to an amount of a RNAi agent sufficient to prevent or ameliorate a disease or one or more symptoms of a disease, including when administered to a subject who has not yet experienced or displayed symptoms of an LPA-associated disease but may be susceptible to the disease. Amelioration of a disease includes slowing the progression of the disease or reducing the severity of subsequent disease. The "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the risk of the disease, and the subject's medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the subject.

[0068] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0069] As used herein, the term "sample" includes similar fluids, cells or tissues separated from a subject, and a collection of fluids, cells or tissues present in the subject. Examples of biological fluids include blood, serum and serosal fluid, blood plasma, cerebrospinal fluid, ocular fluid (ocular fluid), lymph, urine, saliva, etc. Tissue samples can include samples from tissues, organs or local regions. For example, samples can be derived from the fluid or cells in a particular organ, an organ part or these organs. In certain embodiments, samples can be derived from liver (for example, whole liver or some sections of liver, or some types of cells in liver, for example, hepatocytes). In a preferred embodiment, "sample derived from experimenter" refers to blood or blood plasma extracted from this experimenter. In other embodiments, "sample derived from experimenter" refers to liver tissue (or its subcomponent) derived from this experimenter.

[0070] Herein, unless otherwise specified, when referring to any nucleotide position of any strand of an siRNA motif, siRNA modification, siRNA conjugate, siRNA duplex, etc., the 5' to 3' direction is intended.

[0071] In one aspect, the present disclosure provides a siRNA duplex comprising a sense strand and an antisense strand forming a reverse complementary double-stranded region, wherein the antisense strand comprises at least 15, 16, 17, 18 or 19 consecutive nucleotide segments of a sequence as shown in any one of SEQ ID NOs: 122, 131, 148, 151, 152, 171, or a modified segment thereof.

[0072] In some embodiments, the sense strand comprises at least 15, 16, 17, 18, or 19 consecutive nucleotide segments of a sequence as shown in any one of SEQ ID NOs: 13, 22, 39, 42, 43, 62, or a modified segment thereof.

[0073] In some embodiments, the length of the reverse complementary double-stranded region is 17 to 21 bp, such as 20 or 21 bp.

[0074] In some embodiments, the sense strand and antisense strand are independently 19 to 23 nucleotides in length; preferably, the sense strand comprises 19 to 21 nucleotides, and the antisense strand comprises 21 to 23 nucleotides.

[0075] In some embodiments, the siRNA duplex comprises any one or a combination of at least two of the following oligonucleotide duplexes consisting of a sense strand and an antisense strand pairing, optionally wherein the sense strand and the antisense strand each independently comprise at least one modified nucleotide:

[0076] (1) The sense strand has a sequence as shown in SEQ ID NO: 13 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 122 or a fragment thereof, or a modified sequence thereof;

[0077] (2) the sense strand has a sequence as shown in SEQ ID NO: 22 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 131 or a fragment thereof, or a modified sequence thereof;

[0078] (3) the sense strand has a sequence as shown in SEQ ID NO: 39 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 148 or a fragment thereof, or a modified sequence thereof;

[0079] (4) the sense strand has a sequence as shown in SEQ ID NO: 42 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 151 or a fragment thereof, or a modified sequence thereof;

[0080] (5) the sense strand has a sequence as shown in SEQ ID NO: 43 or a fragment thereof, or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 152 or a fragment thereof, or a modified sequence thereof;

[0081] (6) The sense strand has a sequence as shown in SEQ ID NO: 62 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 171 or a fragment thereof, or a modified sequence of the sequence or a fragment thereof.

[0082] In some embodiments, the siRNA duplex is an RNAi agent for inhibiting LPA gene expression.

[0083] In some embodiments, the sense strand differs from any one of SEQ ID NOs: 13, 22, 39, 42, 43, and 62 by 1 to 3 nucleotides.

[0084] In some embodiments, the antisense strand differs from any one of SEQ ID NOs: 122, 131, 148, 151, 152, and 171 by 1 to 3 nucleotides.

[0085] In some embodiments, the sense strand has the same or a different number of nucleotides than the antisense strand.

[0086] In some embodiments, the sense strand has 19 nucleotides and the antisense strand has 19 nucleotides.

[0087] In some embodiments, the sense strand has 19 nucleotides and the antisense strand has 21 nucleotides.

[0088] In some embodiments, the sense strand has 20 nucleotides and the antisense strand has 20 nucleotides.

[0089] In some embodiments, the sense strand has 20 nucleotides and the antisense strand has 22 nucleotides.

[0090] In some embodiments, the sense strand has 21 nucleotides and the antisense strand has 21 nucleotides.

[0091] In some embodiments, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0092] In some embodiments, the sense strand has 23 nucleotides and the antisense strand has 23 nucleotides.

[0093] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0094] In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0095] In some embodiments, the sense strand and antisense strand each independently comprise at least one modified nucleotide.

[0096] In some embodiments, at least one modified nucleotide is selected from any one or a combination of at least two of the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-anhydrohexitol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0097] In some embodiments, all nucleotides on the sense strand and the antisense strand are modified at the 2' position of the ribose sugar of the nucleotide.

[0098] In some embodiments, the modification at the 2' position of the ribose of the nucleotide is selected from any one or a combination of 2'-methoxy modification, 2'-methoxyethyl modification, 2'-fluoro modification, 2'-benzyloxy modification, 2'-methylcarbonylamino modification and 2'-pyridylmethoxy modification.

[0099] In some embodiments, the modification at the 2' position of the ribose sugar of each nucleotide is selected from a combination of a 2'-methoxy modification and a 2'-fluoro modification.

[0100] In some embodiments, the modification at the 2' position of the ribose sugar of each nucleotide is selected from alternating combinations of 2'-methoxy modifications and 2'-fluoro modifications.

[0101] In some embodiments, the 2' position of each nucleotide ribose is modified as follows: the odd-numbered positions of the sense strand are all 2'-fluoro modified, and the even-numbered positions are all 2'-methoxy modified; and the odd-numbered positions of the antisense strand are all 2'-methoxy modified, and the even-numbered positions are all 2'-fluoro modified.

[0102] In some embodiments, the nucleotides are linked to each other via 3',5'-phosphodiester bonds.

[0103] In some embodiments, the nucleotides are linked to each other by 3',5'-phosphothioate diester bonds.

[0104] In some embodiments, the aforementioned oligonucleotides have alternating fluorine and oxygen modifications.

[0105] In some embodiments, the sense strand and / or antisense strand of the modified siRNA has a 3',5'-phosphorothioate diester bond between the first and second nucleotides from the 3' end and / or the 5' end. For example, in some embodiments, a chirally pure 3',5'-phosphorothioate diester bond is formed. In some embodiments, the sense strand and / or antisense strand may contain one, two, or three 3',5'-phosphorothioate diester bonds between the first and fourth nucleotides from the 5' end, and the antisense strand may contain one, two, or three 3',5'-phosphorothioate diester bonds between the first and fourth nucleotides from the 3' end. In some embodiments, there are 3',5'-phosphorothioate bonds between the first and second nucleotides and between the second and third nucleotides from the 5' end of the sense strand; there are 3',5'-phosphorothioate bonds between the first and second nucleotides and between the second and third nucleotides from the 5' end of the antisense strand, and there are 3',5'-phosphorothioate bonds between the first and second nucleotides and between the second and third nucleotides from the 3' end of the antisense strand.

[0106] In some embodiments of the present invention, for example, the 2'-position of the ribose of each nucleotide is modified as follows: the odd-numbered positions of the sense strand are all 2'-fluoro modified, and the even-numbered positions are all 2'-methoxy modified; and the odd-numbered positions of the antisense strand are all 2'-methoxy modified, and the even-numbered positions are all 2'-fluoro modified;

[0107] There are 3',5'-phosphorothioate bonds between the first and second nucleotides and between the second and third nucleotides from the 5' end of the sense strand; there are 3',5'-phosphorothioate bonds between the first and second nucleotides and between the second and third nucleotides from the 5' end of the antisense strand, and there are 3',5'-phosphorothioate bonds between the first and second nucleotides and between the second and third nucleotides from the 3' end of the antisense strand. Examples of modified siRNA duplexes include A01501-AL, A02401-AL, A04101-AL, A04401-AL, A04501-AL, and A06601-AL.

[0108] The present disclosure provides an siRNA conjugate, which comprises the siRNA duplex as described in the present disclosure and a conjugated group connected to the siRNA duplex.

[0109] The double-stranded RNA and double-stranded RNA modifications disclosed herein can be optionally connected to one or more conjugated groups. The conjugated group can be attached to the sense strand, antisense strand, or both strands at the 3' end, 5' end, or both ends. For example, the conjugated group can be connected to the sense strand. In a preferred embodiment, the conjugated group is connected at the 3' end of the sense strand. In one embodiment, the conjugated group has an arbitrary GalNAc structure.

[0110] In some embodiments, the conjugate group is attached at the 3' end or the 5' end of the sense strand of the nucleotide.

[0111] In some embodiments, the conjugate group is one or more GalNAc derivatives attached using a bivalent or trivalent branched linker.

[0112] Typically, the conjugated group includes at least one pharmaceutically acceptable targeting group, or further includes a linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the targeting group is 1 to 6. In some embodiments, the targeting group is 2 to 4. In some embodiments, the targeting group is 3. The conjugated group can be covalently or non-covalently attached to the siRNA molecule, and the attachment site can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the attachment site is at the 3' end of the siRNA sense strand.

[0113] In some embodiments, the pharmaceutically acceptable targeting group may be a conventional ligand in the field of siRNA administration, such as the various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

[0114] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to an asialoglycoprotein receptor (ASGPR) on the liver surface. The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of siRNA connected to the ligand to the target cells.

[0115] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. That is, after the siRNA molecule forms the siRNA conjugate with the conjugated group containing the galactose or N-acetylgalactosamine molecule as the targeting group, the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4. In some embodiments, when the siRNA is linked to the conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0116] The targeting group can be connected to the siRNA molecule via a suitable linker. Those skilled in the art can select a suitable linker based on the specific type of the targeting group. For information on these linkers, the type of targeting group, and the method of connection to the siRNA, please refer to the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0117] In some embodiments, the structure of the conjugate group is, for example:

[0118] ,

[0119] wherein X is a hydroxyl protecting group or H, wherein the hydroxyl protecting group is selected from acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, wherein the amine protecting group is selected from formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer from 0 to 20; and q, r and s are each independently an integer from 1 to 7.

[0120] In some embodiments, the structure of the conjugate group is, for example:

[0121] .

[0122] In some embodiments, the conjugated group is, for example:

[0123]

[0124] wherein X is oxygen, -N(Y)- or sulfur;

[0125] Y is C 1-4 Alkyl or C 6-10 aryl;

[0126] R1 is oxygen or sulfur;

[0127] R2 is hydrogen, -NH2, C 1-4 Alkyl, C 6-10 Aryl, C 1-4 Alkoxy or halogen;

[0128] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0129] B is -(CH2) e -, where e is an integer from 0 to 7;

[0130] L is -CONH- or -NHCO-;

[0131] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0132] X2 is -(CH2) g -, g is an integer from 1 to 6;

[0133] X3 is oxygen or sulfur;

[0134] Y1 is 0 or 1;

[0135] Y2 is 0, 1, or 2;

[0136] When Y3 is 1, X4 is CH2; when Y3 is 2, X4 is CH; when Y3 is 3, X3 is carbon;

[0137] m is an integer from 0 to 4;

[0138] n is an integer from 0 to 4.

[0139] In some embodiments, the conjugate group has, for example, any of the following structures:

[0140] ,

[0141] ,

[0142] ,or

[0143] .

[0144] In some embodiments, the siRNA conjugates of the present disclosure may have any of the following structures:

[0145] ,

[0146] ,

[0147] ,

[0148] ,or

[0149] .

[0150] In some embodiments, the conjugate group has, for example, the following structure:

[0151]

[0152] wherein X is oxygen, -N(Y)- or sulfur;

[0153] Y is C 1-4 Alkyl or C 6-10 aryl;

[0154] R1 is oxygen or sulfur;

[0155] R2 is hydrogen, -NH2, C 1-4 Alkyl, C 6-10 Aryl, C 1-4 Alkoxy or halogen;

[0156] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d -, wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0157] B is -(CH2) e -, where e is an integer from 0 to 7;

[0158] L is -CONH- or -NHCO-;

[0159] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0160] X2 is -(CH2) g -, g is an integer from 1 to 6;

[0161] X3 is oxygen or sulfur;

[0162] Y1 is 0 or 1;

[0163] Y2 is 0, 1, or 2;

[0164] When Y3 is 1, X4 is CH2; when Y3 is 2, X4 is CH; when Y3 is 3, X3 is carbon;

[0165] m is an integer from 0 to 4;

[0166] n is an integer from 0 to 4;

[0167] q is an integer from 0 to 4.

[0168] In some embodiments, wherein the conjugated group has, for example, any of the following structures:

[0169] , , , ,or .

[0170] In some embodiments, the siRNA conjugates of the present disclosure have, for example, any of the following structures:

[0171] ,

[0172] ,

[0173] ,

[0174] ,or

[0175] .

[0176] Wherein, either or both of the sense strand and the antisense strand can be connected to the conjugated group G4, G5, G6, G7, G101, G102, G103, G105 or G106.

[0177] In some embodiments, the conjugate group is attached to the 3' end of the sense strand.

[0178] The present disclosure also provides a nucleic acid-protein complex comprising the double-stranded region or antisense strand of the aforementioned double-stranded RNAi agent or siRNA conjugate, and a nuclease.

[0179] In this disclosure, the term "nucleic acid-protein complex" refers to the RNA-induced silencing complex (RISC) formed by the binding of siRNA to the Argonaute protein (AGO). The siRNA is then unzipped into its sense and antisense strands. The sense strand is degraded, while the antisense strand (guide strand) RISC binds to the target mRNA homologous to the siRNA through base pairing. RISC functions as a nuclease, and siRNA guides RISC to cleave the homologous single-stranded mRNA, rendering the mRNA inoperable and unable to produce protein, effectively silencing the gene.

[0180] The present disclosure also provides a recombinant vector comprising a nucleic acid molecule encoding the siRNA as disclosed.

[0181] In some embodiments, the vector backbone of the recombinant vector is selected from a recombinant viroid-derived circular RNA vector, tRNA, rRNA scaffold, and a chimeric tRNA / pre-mi RNA vector.

[0182] The present disclosure also provides a recombinant cell comprising the aforementioned siRNA or recombinant vector.

[0183] In some embodiments, the recombinant cell is selected from Rhodopseudomonas sulfophilus and RNase III-deficient Corynebacterium glutamicum.

[0184] As used herein, a "recombinant vector" is preferably a vector comprising regulatory sequences operably linked to a nucleotide sequence encoding the sense strand contained in a nucleic acid molecule of the present invention. A "recombinant cell" is a cell into which has been introduced at least one recombinant vector capable of expressing a nucleic acid molecule or at least one chain of such a nucleic acid molecule.

[0185] The present disclosure also provides a method for preparing the siRNA described in the present disclosure, which comprises culturing the aforementioned recombinant cells, or directly obtaining the siRNA by chemical synthesis and mixing.

[0186] The present disclosure also provides a pharmaceutical composition, which includes the siRNA duplex or the corresponding siRNA conjugate, and a pharmaceutically acceptable carrier.

[0187] In one embodiment, provided herein are pharmaceutical compositions comprising an siRNA duplex as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising iRNA can be used to treat or prevent diseases or conditions associated with the expression or activity of the LPA gene, such as atherosclerosis. Such pharmaceutical compositions are formulated based on the delivery model. One example is a composition formulated for systemic administration via parenteral delivery, such as subcutaneous (SC) injection. Another example is a composition formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, such as via continuous pump infusion.

[0188] Pharmaceutical compositions comprising the RNAi agents of the present disclosure can be, for example, solutions with or without a buffer or compositions containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micellar formulations, emulsions, and gene therapy vectors.

[0189] In the method of the present disclosure, the siRNA can be administered in a solution. A free siRNA can be administered in a non-buffered solution, for example, in physiological saline or in water. Alternatively, the free siRNA can also be administered in a suitable buffer solution. The buffer solution can include acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and volume molar osmotic pressure concentration of the buffer containing the siRNA can be adjusted so that it is suitable for administering to a subject.

[0190] In some embodiments, the buffer solution further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, such as the physiological value of human plasma. Solutes that can be added to the buffer solution to control osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0191] The pharmaceutical compositions of the present disclosure can be administered at a dose sufficient to inhibit the expression of the LPA gene. Typically, a suitable dose of the siRNA of the present disclosure is in the range of about 0.001 to about 200.0 mg per kilogram of body weight of the recipient per day, typically in the range of about 1 to 50 mg per kilogram of body weight per day. For example, siRNA (e.g., siRNA conjugate) can be administered at a dose of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3. 3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7 .6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 48, 49, or about 50 mg / kg.

[0192] The pharmaceutical composition can be given once a day, or given repeatedly at different time intervals of 1 to 365 days, or the siRNA can be given twice, three times or more subdose at appropriate intervals within one year, or even can be given by continuous infusion or delivery through a controlled release formulation. In this case, the siRNA contained in each subdose must be correspondingly less, so as to realize a daily total dose. Dosage unit compounding can also be used to deliver in a few days, for example, using a conventional sustained release formulation that provides a lasting siRNA release within a few days' timeframe. Sustained release formulations are well known in the art and are particularly useful for delivering reagents at specific sites, and can be used with reagents of the present disclosure thus. In this embodiment, the dosage unit comprises corresponding multiple daily doses.

[0193] In other embodiments, a single dose of the pharmaceutical composition can be long-lasting, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once per week. In other embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once every two months.

[0194] Those skilled in the art will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the subject's overall health and / or age, and other existing diseases. In addition, treating a subject with a therapeutically effective dose of a composition may include a single treatment or a series of treatments. As described elsewhere herein, the effective dosage and in vivo half-life of each siRNA encompassed by the present disclosure may be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0195] The pharmaceutical compositions of the present disclosure can be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a skin patch); pulmonary; e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer; intratracheal; intranasal; epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implant device; or intracranial, e.g., intraparenchymal, intrathecal, or intraventricular administration.

[0196] The siRNA used in the compositions and methods of the present disclosure can be formulated to deliver, for example, liposomes or micelles in a membrane molecule assembly. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one lipid bilayer (e.g., one lipid bilayer or multiple lipid bilayers), having an outer membrane formed by a lipophilic material and an aqueous portion located inside. The lipophilic material separates the aqueous interior from the aqueous exterior (although in some instances, it may include) that typically does not include the siRNA composition. Liposomes are useful for transferring active ingredients and delivering them to the site of action. Because the liposome membrane is structurally similar to a biological membrane, when liposomes are applied to a tissue, the liposome bilayer fuses with the bilayer of the cell membrane. As the fusion of liposomes and cells proceeds, the internal aqueous contents including siRNA are delivered to the cell, wherein the siRNA can specifically bind to a target RNA and can mediate RNA interference (RNA interference, RNAi). In some cases, these liposomes are also specifically targeted, such as guiding the siRNA to a specific cell type.

[0197] Liposomes comprising siRNA can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The siRNA formulation is then added to the micelle comprising the lipid component. The cationic group on the lipid interacts with the siRNA and condenses around the siRNA to form a liposome. After condensation, the detergent is removed, for example, by dialysis to obtain the corresponding liposome formulation of the siRNA.

[0198] siRNA, such as the RNA duplexes of the present disclosure, can be encapsulated in lipid formulations (eg, LNPs or other nucleic acid-lipid particles).

[0199] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNP contains a cationic lipid, a non-cationic lipid, and a lipid (e.g., a PEG-lipid conjugate) that stops the particle aggregation. LNP is extremely useful for synthetic applications because they demonstrate a circulation life that is extended after intravenous (iv) injection and accumulate at distal sites (e.g., at a site physically separated from the site of administration).

[0200] In one embodiment, the mass ratio of lipid to siRNA duplex is about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0201] In some preferred embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structured lipids, and polymer-conjugated lipids.

[0202] In some preferred embodiments, the cationic lipid is a compound of formula (I), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight chain alkyl; L2 is C 12～25 Branched alkyl, for example, YK-009 of formula (II) (see patent CN114044741B, the entire contents of which are incorporated herein by reference, especially including the general formula and specific compounds therein).

[0203] (I)

[0204] (II)

[0205] In some preferred embodiments, the cationic lipid is a compound of formula (II), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein: G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 Straight or branched alkyl; R2 is C 6~25 A linear or branched alkyl group; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; and L is (CH2)2- or -(CH2)3- or -(CH2)4-. For example, YK-401 of formula (II-I), YK-402 of formula (II-II), and YK-407 of formula (II-III) (see patent CN115784921B, the entire contents of which are incorporated herein by reference, particularly including the general formula and specific compounds therein).

[0206] (II)

[0207] (II-I)

[0208] (II-II)

[0209] (II-III)

[0210] In some preferred embodiments, the cationic lipid is a compound of formula (III), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein: G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight or branched alkyl; R2 is C 12~25 Branched alkyl group; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-. For example, YK-201 of formula (III-I), YK-202 of formula (III-II), etc. (see patent CN115677518B, the entire contents of which are incorporated herein by reference, especially including the general formula and specific compounds therein).

[0211] (III)

[0212] (III-I)

[0213] (III-II)

[0214] In some preferred embodiments, the cationic lipid is a compound of formula (IV), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight or branched alkyl; R2 is C 12~25 Straight or branched alkyl; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3 is -CH3 or -CH2CH3 or -CH2CH2OH For example, YK-305 of formula (IV-I), YK-310 of formula (IV-II), etc. (see patent CN115745820B, the entire contents of which are incorporated herein by reference, especially including the general formula and specific compounds therein).

[0215] (IV)

[0216] (IV-I)

[0217] (IV-II)

[0218] In some preferred embodiments, the cationic lipid is a compound of formula (V), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein G 1 and G 2 Each independently is an unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 Alkenyl; R 3 OR 5 、N、-C(=O)OR 4 、-OC(=O)R 4 or -NR 5 C(=O)R 4 ; R 4 C1-C 12 hydrocarbon group; and R 5 is H or a C1-C6 hydrocarbon group; for example, ALC0315 of formula (VI) (see patent CN108368028B, the entire contents of which are incorporated herein by reference, especially including the general formula and specific compounds therein);

[0219] (V)

[0220] (VI)

[0221] In some preferred embodiments, the cationic lipid is a compound of formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, wherein R4 is selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of: -OR, -OH, -O(CH2)n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocycle; n is 1, 2 or 3; for example, SM102 of formula (VI-I) (see patent application CN110520409A, the entire contents of which are incorporated herein by reference, especially including the general formula and specific compounds therein).

[0222] (VI)

[0223] (VI-I)

[0224] In some preferred embodiments, the cationic lipid is a compound of formula (VII), or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof (see patent CN102625696B, DLIN-MC3-DMA, the entire contents of which are incorporated herein by reference, especially including the general formula and specific compounds therein, etc.),

[0225] (VII).

[0226] In some more preferred embodiments, the cationic lipid is selected from any one or a combination of at least two of the group consisting of YK-009, YK-401, YK-305, ALC0315, SM102 and DLIN-MC3-DMA.

[0227] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0228] In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0229] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-70):(0.5-5).

[0230] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25-65):(5-25):(25-45):(0.5-5).

[0231] In some more preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0232] In some preferred embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterol and derivatives thereof.

[0233] In some more preferred embodiments, the neutral lipid is selected from any one or a combination of at least two of the following groups: 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dicondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-Di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0234] In some preferred embodiments, the structured lipid is selected from any one or a combination of at least two of the following groups: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0235] In some preferred embodiments, the polymer-conjugated lipid is selected from any one or a combination of at least two of the following groups: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

[0236] In some more preferred embodiments, the polymer-conjugated lipid is selected from any one or a combination of at least two of the following groups: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000) and methoxypolyethylene glycol ditetradecanoyl acetamide (ALC-0159).

[0237] Examples of pharmaceutical compositions of the present disclosure include, but are not limited to, aqueous formulations, emulsion formulations, and liposome-containing formulations. These compositions can be produced from a variety of components, examples of which include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. For example, formulations that target the liver when treating liver disorders (e.g., liver cancer) are preferred.

[0238] The pharmaceutical formulations of the present disclosure (which may conveniently be in unit dosage form) can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the steps of combining the active ingredients with the pharmaceutical carrier(s) or excipient(s). Generally speaking, the formulations are prepared by uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier or both, and, if desired, shaping the product.

[0239] Compositions of the present disclosure can be formulated as any one of many possible dosage forms, such as but not limited to tablets, capsules, gelatin capsules, liquid syrups, soft capsules, suppositories and enemas. Compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further include materials that increase the viscosity of the suspension, such materials including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also include stabilizers.

[0240] Certain compositions of the present disclosure also incorporate a carrier compound into the formulation. As used herein, a "carrier compound" or "carrier" may refer to a nucleic acid or its analog that is inert (i.e., not biologically active per se) but is recognized as a nucleic acid during in vivo processes, such as by degrading the biologically active nucleic acid or promoting its removal from the circulation, thereby reducing the bioavailability of the biologically active nucleic acid. Co-administration of a nucleic acid and a carrier compound (generally in excess of the latter) can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidneys, or other external circulation reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of partially phosphorothioated dsRNA in liver tissue can be reduced when co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4'isothiocyanatostilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0241] In contrast to carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components of a particular pharmaceutical composition, with reference to the intended mode of administration. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0242] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration, which do not react toxicly with nucleic acids, can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like.

[0243] The preparation that is used for local administration of nucleic acid can comprise the aseptic or non-sterile aqueous solution, the non-aqueous solution in common solvent such as alcohol, or the nucleic acid solution in liquid or solid oil matrix.These solutions can also comprise buffer, diluent and other suitable additives.Can use be suitable for non-parenteral administration and not with nucleic acid generation toxic reaction, pharmaceutically acceptable organic or inorganic excipient.

[0244] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0245] The present disclosure also provides methods for treating or preventing diseases and conditions that can be modulated by downregulating LPA gene expression, such as 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 associated with elevated levels of Lp(a) particles, as well as other related conditions, pathologies or syndromes not yet identified.

[0246] The siRNAs of the present invention can be administered to a subject using any mode of administration known in the art, including but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof. In a preferred embodiment, the agents are administered subcutaneously.

[0247] In other embodiments, the siRNA is administered in combination with another therapeutic agent. The siRNA and the additional therapeutic agent can be administered in combination in the same composition, for example, parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

[0248] Examples of additional therapeutic agents include agents known to treat LPA-related diseases or conditions. For example, additional therapeutic agents include: administering one or more siRNAs of the present invention to a subject; administering a non-LPA RNAi therapeutic to a subject; and modifying a behavior in a subject. In some embodiments, the non-LPA RNAi therapeutic is an additional therapeutic agent selected from the group consisting of HMg Co-A reductase inhibitors (statins), ezetimibe, PCSK-9 inhibitors, CTEP inhibitors, ANGPTL3-targeted therapies, APOC3-targeted therapies, and niacin, or any combination thereof.

[0249] In one embodiment, the iRNA agent is administered to a patient and subsequently the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered simultaneously.

[0250] The application scenarios of "non-diagnostic or non-therapeutic purposes" described in the present disclosure include but are not limited to: using it as a positive control to screen other siRNA duplexes that inhibit LPA gene expression, or using it as an inhibition method in the laboratory to study the relationship between abnormal LPA gene expression and disease.

[0251] The nucleotide codes in this article are shown in Table 1 below:

[0252] Table 1

[0253]

[0254] Example

[0255] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although indicating specific embodiments of the present disclosure) are given for illustrative purposes only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art after reading the detailed description.

[0256] The experimental techniques and methods used in the present examples are conventional techniques unless otherwise specified. For example, in the following examples, where specific conditions are not specified, conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer are generally followed. Materials and reagents used in the examples are commercially available unless otherwise specified.

[0257] Those skilled in the art will understand that in various embodiments of the present disclosure, when the test substance in the experiment is a siRNA conjugate, it includes but is not limited to inhibition rate, IC 50 、IC 40 The experimental data and results can reflect the inhibition rate, IC 50 、IC 40 For those skilled in the art, there is no difficulty in understanding this.

[0258] Example 1: Inhibitory effect of siRNA motif on LPA gene

[0259] Based on the human LPA mRNA sequence (NM_005577.4), 110 siRNA motifs were designed and synthesized, as shown in Table 2. The inhibitory effect of each siRNA motif on LPA gene expression was detected by the dual luciferase system, as shown in Table 4.

[0260] 1.1 Synthesis of siRNA Motifs

[0261] Instruments and reagents: Qingke 192 P automatic DNA / RNA synthesizer, whose solid phase support is a universal support of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0262] Preparation method:

[0263] The following nucleotide monomer solutions were prepared using acetonitrile at a monomer concentration of 0.15 M: DMT-A-2'-O-TBDMS phosphoramidite monomer (Formula 9), DMT-C-2'-O-TBDMS phosphoramidite monomer (Formula 10), DMT-G-2'-O-TBDMS phosphoramidite monomer (Formula 11), and DMT-U-2'-O-TBDMS phosphoramidite monomer (Formula 12).

[0264] Formula 9 Formula 10

[0265] Formula 11 Formula 12

[0266] By solid phase phosphoramidite method, nucleoside monomers are connected one by one in the 3'-5' direction according to the nucleotide arrangement order. Each connection of a nucleoside monomer includes a four-step reaction of deprotection, coupling, oxidation or sulfidation, and hydroxyl protection. Wherein, when two nucleotides are connected by phosphate ester, when connecting the latter nucleoside monomer, the four-step reaction of deprotection, coupling, oxidation, and hydroxyl protection is included. When two nucleotides are connected by phosphorothioate, when connecting the latter nucleoside monomer, the four-step reaction of deprotection, coupling, sulfidation, and hydroxyl protection is included.

[0267] Specifically prepared by the following steps:

[0268] The solid phase carrier is loaded into the designated position of the synthesizer, and the corresponding product is obtained after several synthesis cycles. The synthesis cycle includes (1) deprotection, (2) coupling, (3) oxidation / sulfurization and (4) hydroxyl protection. The cycle process and the reagents used are described as follows:

[0269] (1) Deprotection

[0270] 3% dichloroacetic acid in toluene was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0271] (2) Coupling

[0272] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolium as an activating agent, followed by acetonitrile washing.

[0273] (3) Oxidation / sulfurization

[0274] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (90 / 10) as an oxidant, followed by rinsing with acetonitrile.

[0275] Sulfurization: Use 3% hydrogenated xanthan gum in pyridine as the sulfurizing agent for sulfurization, and then use acetonitrile for washing.

[0276] (4) Hydroxyl protection

[0277] Hydroxyl protection was performed using 10% acetic anhydride in tetrahydrofuran (CAP A) or tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting reagents, followed by rinsing with acetonitrile.

[0278] Repeat the above operation and cycle through the above steps according to the set nucleotide arrangement order to obtain a sense chain product or an antisense chain product with a specific sequence arrangement.

[0279] (5) Use 3% dichloroacetic acid toluene solution as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile for washing.

[0280] (6) Ammonolysis and purification

[0281] The solid phase support after the reaction was transferred to a reactor, and concentrated ammonia water (25-28%, mass percentage) was added. After maintaining the aminolysis at 60°C for 12 hours, the system was cooled to room temperature, and the mixture was filtered. The filter cake was rinsed with a mixed solution of purified water and ethanol. The filtrate was combined, passed through a chromatography column, concentrated, and freeze-dried to obtain a 2'-O-TBDMS-protected product.

[0282] (7) Remove TBDMS

[0283] Add DMSO and triethylamine hydrofluoric acid to the resulting product and react at 60°C for 2 hours. Then, add ammonium acetate aqueous solution to the reaction solution, shake and mix thoroughly, add anhydrous ethanol, shake and mix thoroughly, and crystallize at -20°C for 8-12 hours. After centrifugation, discard the supernatant and rinse the precipitate with anhydrous ethanol to obtain the unmodified single-chain product.

[0284] (8) Annealing

[0285] The sense and antisense strands of the obtained siRNA motif were mixed in a 1:1 molar ratio, heated to 95°C and maintained for 3 minutes, and then slowly cooled to room temperature to form a double-stranded siRNA motif.

[0286] The 110 siRNA motifs in Table 2 were synthesized according to the above method, wherein APC is a positive control, which is the unmodified base sequence of Amgen's drug Olpasiran, and ANC is a negative control, which is an unmodified nonsense sequence.

[0287] Table 2 siRNA motifs

[0288]

[0289]

[0290]

[0291] 1.2 Dual luciferase assay to detect the inhibitory effect of siRNA motifs on the LPA gene

[0292] The experimental materials and methods are as follows:

[0293] 1.2.1 Experimental Materials

[0294] Table 3

[0295]

[0296] psiCHECK2-LPA plasmid construction: The full-length mRNA sequence of the LPA gene (NM_007755.4) was cloned into the dual-luciferase plasmid psiCHECK TM -2, commissioned by Yunzhou Biotechnology (Guangzhou) Co., Ltd., catalog number VB240227-1779ggq.

[0297] 1.2.2 Experimental methods

[0298] 1) Plasmid transfection and cell plating

[0299] Day 0: Transform psiCHECK2-LPA plasmid into Huh7 cells

[0300] Dilute the psiCHECK2-LPA plasmid to 10 ng / μL with Opti-MEM. Take Huh7 cells, wash them with DPBS, add trypsin for digestion, and adjust the cell density to 1×10 5 cells / mL. Mix Fugene-HD transfection reagent and 10 ng / μL psiCHECK2-LPA plasmid dilution at a ratio of 3:100 (volume ratio). Incubate at room temperature for 10 minutes. After incubation, add the solution to Huh7 cells and seed them into a 96-well plate at a density of 10,000 cells per well in 100 μL of culture medium per well. Culture Huh7 cells overnight in a 5% CO2, 37°C incubator.

[0301] 2) Preparation of siRNA solution and cell transfection

[0302] Day 1: siRNA treatment

[0303] RNAiMAX transfection reagent and Opti-MEM were mixed at a volume ratio of 1.5:48.5 to obtain Mixture X. Incubate at room temperature for 15 minutes. Test siRNA solutions with starting concentrations of 12 nM and 60 nM were mixed with Mixture X at a volume ratio of 1:1 to obtain Mixture Y. Incubate at room temperature for 15 minutes. After incubation, 20 μL of Mixture Y was added to 100 μL of fresh DMEM at a volume ratio of 1:5 and mixed thoroughly to obtain Mixture Z. The final transfection concentrations of the test siRNAs were 1 nM and 5 nM, respectively. Discard the supernatant from the Huh7 cells cultured overnight in the 96-well plate from step 1. Add 120 μL of Mixture Z to each well of the 96-well plate. The 96-well plate was then incubated in a CO2 cell incubator for 48 hours.

[0304] 3) Cell sampling and fluorescence expression detection

[0305] Day 3: Detection of reporter genes

[0306] Reagent preparation:

[0307] Reagent A: Prepare Dual-Glo® Luciferase Reagent by transferring the contents of one bottle of Dual-Glo® Luciferase Buffer to one bottle of Dual-Glo® Luciferase Substrate. Aliquot and store at -80°C in a dark freezer.

[0308] Reagent B: Calculate the volume of Dual-Glo® Stop & Glo® Reagent required for the experiment. Prepare the required volume of Dual-Glo® Stop & Glo® Reagent using a fresh container using a 1:100 volume ratio of Dual-Glo® Stop & Glo® Substrate to Dual-Glo® Stop & Glo® Buffer. This reagent should be prepared immediately.

[0309] Sample addition and detection:

[0310] Remove the cell supernatant with a pipette, add 75 μL of fresh DMEM medium containing 10% FBS (fetal bovine serum) to each well, equilibrate to room temperature, add 75 μL of reagent A to each well, and shake on a plate shaker at room temperature for 10 minutes to lyse the cells.

[0311] After complete cell lysis was observed under a microscope, the luminescence of firefly luciferase (Firefly lum) was measured on a multi-function microplate reader. The microplate reader parameters were set to chemiluminescence Lum detection, full wavelength, integration time of 1 second, and detection height of 1 mm.

[0312] After the assay is complete, add 75 μL of Reagent B to each well of the plate from the previous step. Shake at room temperature for 10 minutes and measure Renilla luciferase luminescence. Set the microplate reader to chemiluminescence Luminescence detection, full wavelength, 1-second integration time, and 1-mm detection height.

[0313] A control group was set up in the experiment, in which Opti-MEM was used instead of the above siRNA solution, and the other conditions were the same as those of the experimental group; a blank group consisted of Huh7 cells that were not transfected with the psiCHECK2-LPA plasmid and to which no siRNA was added.

[0314] 4) Calculate the inhibition rate of siRNA on target gene

[0315] The ratio of the fluorescence value of Renilla luciferase to that of Firefly luciferase is recorded as α, and the calculation formula is:

[0316] α = (average value of Renilla lum in test wells - average value of Renilla lum in blank control group) / (average value of Firefly lum in test wells - average value of Firefly lum in blank control group);

[0317] The experimental group ratio calculated according to the above formula is recorded as: α (experimental group), and the control group ratio is recorded as: α (transfection reagent control group).

[0318] The inhibition rate of siRNA against target gene expression was calculated according to the following formula:

[0319] Inhibition rate (%) = [1-α (average value of experimental group) / α (average value of transfection reagent control group)] × 100%.

[0320] Three parallel detection wells were set up for each of the two transfection concentrations. The inhibition rate of each siRNA motif on LPA mRNA expression is shown in Table 4 below.

[0321] Table 4 Inhibition rate of siRNA motif on LPA gene (%)

[0322]

[0323]

[0324] Example 2: Synthesis of Alternately Modified siRNA Modifiers

[0325] To improve the inhibition rate and stability, the siRNA motifs in Table 2 were modified with alternating 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) groups, and 3',5'-phosphorothioate bonds were present between the nucleotides at the 5' and / or 3' ends.

[0326] 2.1 Synthesis of alternatively modified siRNAs

[0327] The rule of alternating modification in the present disclosure is: the nucleotides at odd-numbered positions of the sense chain and the nucleotides at even-numbered positions of the antisense chain are all modified with 2'-F, and the nucleotides at other positions are modified with 2'-OMe. In addition, the alternatingly modified siRNA modifications in the present disclosure have 3',5'-phosphorothioate bonds between the first and second nucleotides at the 5' end of the sense chain and between the second and third nucleotides; the antisense chain has 3',5'-phosphorothioate bonds between the first and second nucleotides at the 5' end and between the second and third nucleotides, and the antisense chain has 3',5'-phosphorothioate bonds between the first and second nucleotides at the 3' end and between the second and third nucleotides. The alternatingly modified siRNA modifications in the present disclosure are indicated by adding "-AL" after the number of their corresponding basic sequences, as shown in Table 5.

[0328] Instruments and reagents: Qingke 192 P automatic DNA / RNA synthesizer, whose solid phase support is a universal support of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0329] For example, the preparation method may include:

[0330] The following nucleotide monomer solutions were prepared in acetonitrile at a monomer concentration of 0.15 M: DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3), DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-AF phosphoramidite monomer (Formula 5), DMT-CF phosphoramidite monomer (Formula 6), DMT-GF phosphoramidite monomer (Formula 7), and DMT-UF phosphoramidite monomer (Formula 8).

[0331]

[0332]

[0333]

[0334] By solid phase phosphoramidite method, nucleoside monomers are connected one by one in the 3'-5' direction according to the nucleotide arrangement order. Each connection of a nucleoside monomer includes a four-step reaction of deprotection, coupling, oxidation or sulfidation, and hydroxyl protection. Wherein, when two nucleotides are connected by phosphate ester, when connecting the latter nucleoside monomer, the four-step reaction of deprotection, coupling, oxidation, and hydroxyl protection is included. When two nucleotides are connected by phosphorothioate, when connecting the latter nucleoside monomer, the four-step reaction of deprotection, coupling, sulfidation, and hydroxyl protection is included.

[0335] (1) Deprotection

[0336] 3% dichloroacetic acid in toluene was used as a deprotection agent to remove the DMT protecting group, followed by washing with acetonitrile.

[0337] (2) Coupling

[0338] Each nucleotide monomer was coupled in acetonitrile using 0.25 M 5-ethylthiotetrazolium as an activating agent, followed by acetonitrile washing.

[0339] (3) Oxidation / sulfurization

[0340] Oxidation: Oxidation was performed using a 0.05 M iodine solution in pyridine / water (90 / 10) as an oxidant, followed by rinsing with acetonitrile.

[0341] Sulfurization: Use 3% hydrogenated xanthan gum in pyridine as the sulfurizing agent for sulfurization, and then use acetonitrile for washing.

[0342] (4) Hydroxyl protection

[0343] Hydroxyl protection was performed using 10% acetic anhydride in tetrahydrofuran (CAP A) or tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as hydroxyl protecting reagents, followed by rinsing with acetonitrile.

[0344] Repeat the above steps according to the set nucleotide arrangement order to obtain a sense strand product or an antisense strand product with a specific sequence arrangement.

[0345] (5) Use 3% dichloroacetic acid toluene solution as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile for washing.

[0346] (6) Ammonolysis and purification

[0347] The solid phase support was transferred to a reactor, and concentrated ammonia water (25%-28%, mass percentage) was added. After aminolysis at 60°C for 12 hours, the system was cooled to room temperature, and the mixture was transferred to a filter press for filtration. The filter cake was rinsed with a mixed solution of purified water and ethanol. The filtrates were combined, passed through a chromatography column, concentrated, and freeze-dried to obtain 2'-OMe and 2'-F modified single-chain products.

[0348] (7) Annealing

[0349] The purified sense and antisense strands were mixed in a 1:1 molar ratio, heated to 95°C for 3 min, and then slowly cooled to room temperature to form siRNA duplexes.

[0350] The sense and antisense strands of the siRNAs listed in Table 5 were synthesized according to the above method, totaling 110 siRNAs. APC-AL served as a positive control, consisting of the original sequence of Amgen's Olpasiran (its base sequence is shown in Table 2) with the aforementioned alternating modifications. APC-OL served as a positive control, consisting of the original sequence of Amgen's Olpasiran (its base sequence is shown in Table 2) with the original modifications (the modified sense strand sequence is shown in SEQ ID NO: 219, and the base antisense strand sequence is shown in SEQ ID NO: 220). ANC-AL served as a negative control (its base sequence is shown in Table 2), consisting of an alternatively modified nonsense sequence.

[0351] APC-OL:

[0352] Sense strand (SEQ ID NO: 219): Cms-Ams-Gm-Cm-Cm-Cm-Cm-Um-Uf-Af-Uf-Um-Gm-Um-Um-Am-Um-Am-Cm-Gms-d'A

[0353] Antisense strand (SEQ ID NO: 220): Ums-Cfs-Gm-Uf-Am-Uf-Am-Am-Cm-Am-Am-Uf-Am-Af-Gm-Gf-Gm-Gf-Cms-Ufs-Gm

[0354] Wherein d'A means that the nucleotide is connected to the 20th nucleotide via a 3'-3' phosphodiester bond in DNA.

[0355] Example 3: Inhibitory effect of alternatively modified siRNA modifiers on the LPA gene

[0356] The dual luciferase method and human primary hepatocyte transfection method were used to determine the inhibitory effect of the alternatively modified siRNA modified products synthesized in Example 2 on the LPA gene.

[0357] 3.1 Dual luciferase assay

[0358] The dual luciferase assay was used to detect the inhibitory effects of the 110 siRNA modifications in Table 5 on the LPA gene. The experimental materials and methods were similar to those in 1.2.1 and 1.2.2 of Example 1.

[0359] 3.1.1 Experimental Results

[0360] The inhibitory effects of the siRNA modifications listed in Table 5 on the LPA gene were determined using the method described in 1.2 of Example 1. The inhibition rates of the siRNA modifications at concentrations of 1 nM and 5 nM are shown in Table 5 below. Forty-one of the siRNA modifications exhibited at least 50% inhibition of LPA gene expression at 5 nM. At 1 nM, 21 of the siRNA modifications exhibited inhibition rates higher than that of Pseudomonas aeruginosa. For example, A01501-AL exhibited an inhibition rate of 47.9%, A02401-AL exhibited an inhibition rate of 40.8%, A06601-AL exhibited an inhibition rate of 38.5%, A04501-AL exhibited an inhibition rate of 35.6%, and Pseudomonas aeruginosa APC-AL exhibited an inhibition rate of 35.2%.

[0361] Table 5 Inhibition rate of LPA gene by alternatively modified siRNAs (%)

[0362]

[0363]

[0364] 3.2 Human primary hepatocyte transfection assay to detect the inhibitory effect of alternatively modified siRNAs on the LPA gene

[0365] 19 modified siRNAs with better activity were selected from the 110 modified siRNAs in Table 5 (see Table 7). After the modified siRNAs were transfected into human primary hepatocytes via lipofectamine RNAiMAX, the inhibition rate of each sequence on the LPA gene was detected by qPCR, and the IC was calculated by fitting the dose-effect curve. 40 The siRNA transfection concentration was set to start at 20 nM, and 5-fold gradient dilution was used to prepare 6 concentrations (20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and 0.006 nM) of siRNA working solutions to detect the inhibitory activity against the LPA gene.

[0366] 3.2.1 Experimental Materials

[0367] Table 6

[0368]

[0369] Solvent: sterilized RNase-free water.

[0370] 3.2.2 Experimental methods

[0371] 1) Recovery of primary human hepatocytes

[0372] Preheat the culture medium at 37°C for at least 30 minutes. Add 120 μL of PBS to each well of a collagen-coated 96-well plate, shake several times, and then aspirate the PBS. Remove the cryovial from the liquid nitrogen tank and place it in a 37°C water bath. Gently shake the tube until only a few ice crystals remain. Pour the cell suspension into the recovery medium all at once. Rinse the inner wall of the cryovial with 1 mL of recovery medium two to three times. Invert the cell suspension in the recovery medium to mix thoroughly. Centrifuge at 150 g for 5 minutes at room temperature. Discard the supernatant and resuspend the cells in prewarmed plating medium. Count the cells using a cell counter.

[0373] 2) Plating of primary human hepatocytes and siRNA transfection

[0374] Preparation of siRNA transfection complex: Mix RNAiMAX transfection reagent and Opti-medium in a volume ratio of 1.5:23.5 to obtain solution A, and incubate at room temperature for 15 minutes. Prepare a solution of siRNA modifiers by diluting with Opti-MEM according to the transfection concentration, so that the prepared modifier concentration is 20 times the final transfection concentration. The diluted solution is solution B. Mix solution A and solution B in a 1:1 ratio to obtain solution C. After incubation at room temperature for 15 minutes, add 10 μL to each well of a collagen-coated 96-well cell culture plate.

[0375] Cell dilution and plating: After counting the PHH cells, add plating medium to the cell suspension according to the counting results and adjust the cell concentration to 5×10 5 ~6×10 5 Use a multichannel pipette to dispense the cell suspension into the above collagen-coated 96-well cell culture plate, and add 90 μL of cell suspension to each well.

[0376] Culture: Place the culture plate in a 5% CO2 incubator, 95% relative humidity, and incubate at 37°C for 48 h.

[0377] 3) RNA extraction and reverse transcription

[0378] 24 hours after transfection, the culture medium was removed and the cells were harvested for RNA extraction. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN-74106) according to the kit instructions. cDNA was then synthesized using the FastKing RT Kit (with gDNase) (TIANGEN-KR116-02) according to the kit instructions.

[0379] 4) RT-qPCR

[0380] According to the kit instructions, TaqMan Fast Advanced Master Mix (Thermo, 4444557) was used to amplify the mRNA of the target gene human LPA gene (Thermo, Hs00916691_m1) and the internal reference gene human GAPDH gene (Thermo, Hs02786624_g1).

[0381] 5) Data Analysis

[0382] The ΔΔCt relative quantification method was used to calculate the target gene RNA expression level in each sample based on the Ct value of each sample. The relative expression of the target gene was calculated using 2 -ΔΔCt express.

[0383] The calculation formula is as follows:

[0384] ΔCT = average Ct value of target gene - average Ct value of reference gene;

[0385] ΔΔCT = ΔCT (drug-treated group) - ΔCT (RNAiMAX control group);

[0386] Relative expression of target gene mRNA = 2 -ΔΔCt

[0387] Inhibition rate = (1 – relative expression level of sample / average expression level of RNAiMAX control) × 100%

[0388] GraphPad Prism software (Nonlinear four parameter logistic equations) was used to fit the dose-effect curve.

[0389] 3.2.3 Experimental Results

[0390] After three qPCR tests, the inhibition rates of each siRNA modification on the LPA gene in human primary hepatocytes at different concentrations are shown in Table 7 below. Among them, A04101-AL, A06601-AL, A04501-AL, A01501-AL, A02401-AL, and A04401-AL, a total of 6 siRNA modifications, produced an inhibition rate of more than 40% on LPA, among which A01501-AL had an inhibition rate of 82.6% on LPA at 20 nM. In addition, the IC values of the above 6 siRNA modifications were 40 Better than Yangshen APC-AL, such as B04101-AL's IC 40 It is 0.036 nM, which is better than 7.461 nM of Yangshen APC-AL.

[0391] Table 7 Inhibitory rate and IC of different concentrations of alternating modifiers on LPA in human primary hepatocytes 40

[0392]

[0393] Example 4: Comparison of the inhibition rate of the siRNA motif disclosed herein and similar sequences in the prior art

[0394] In this example, six siRNA motifs A01501, A02401, A04101, A04401, A04501, and A06601 disclosed herein were selected to compare their inhibition rates against the LPA gene with similar sequences in the prior art.

[0395] 1. Experimental Materials

[0396] Test sample: Using the synthesis method of 1.1 in Example 1, the siRNA motif disclosed in the prior art in Table 8 was synthesized.

[0397] Table 8

[0398]

[0399] 2. Experimental methods and results

[0400] The experimental materials and experimental methods were as described in 1.2.1 and 1.2.2 of Example 1, and the siRNA transfection concentration was 5 nM.

[0401] The experimental results are shown in Table 9.

[0402] Table 9

[0403]

[0404] As shown in the table above, the inhibition rates of the six base sequences disclosed herein (i.e., A01501, A02401, A04101, A04401, A04501, and A06601) were compared with similar motifs from the prior art. The results showed that, under the same concentration conditions, the six base sequences disclosed herein all exhibited superior inhibition rates against LPA mRNA expression in Huh7 cells compared to similar sequences from the prior art. For example, compared to the similar sequence A04101P2 disclosed in the prior art, A04101 of the present disclosure exhibited a 76.3% increase in inhibition rate against the LPA gene; compared to the similar sequence A01501P, A01501 of the present disclosure exhibited a 42.6% increase in inhibition rate against the LPA gene.

Claims

1. A siRNA duplex, characterized in that The siRNA duplex comprises a sense strand and an antisense strand forming a reverse complementary double-stranded region, wherein the antisense strand comprises a sequence as shown in any one of SEQ ID NOs: 122, 131, 148, 151, 152, and 171, or a modified sequence thereof; The odd-numbered positions of the modified sequence of the antisense chain are all 2'-methoxy modified, and the even-numbered positions are all 2'-fluoro modified; the modified sequence of the antisense chain is connected by a 3',5'-phosphorothioate bond between the first and second nucleotides and between the second and third nucleotides starting from the 5' end, and is connected by a 3',5'-phosphorothioate bond between the first and second nucleotides and between the second and third nucleotides starting from the 3' end.

2. The siRNA duplex according to claim 1, wherein The sense strand comprises a sequence as shown in any one of SEQ ID NOs: 13, 22, 39, 42, 43, and 62, or a modified sequence thereof; wherein the odd-numbered positions of the modified sequence of the sense strand are all 2'-fluoro modified, the even-numbered positions are all 2'-methoxy modified, and the first and second nucleotides and the second and third nucleotides starting from the 5' end of the modified sequence of the sense strand are connected by a 3',5'-phosphorothioate bond.

3. The siRNA duplex according to claim 1, wherein The siRNA duplex is an RNAi agent for inhibiting LPA gene expression.

4. The siRNA duplex according to claim 2, wherein The siRNA duplex comprises any one or a combination of at least two selected from the following oligonucleotide duplexes consisting of sense and antisense strand pairs: (1) The sense strand has the sequence shown in SEQ ID NO: 13 or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO: 122 or a modified sequence thereof; (2) the sense strand has the sequence shown in SEQ ID NO: 22 or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO: 131 or a modified sequence thereof; (3) the sense strand has the sequence shown in SEQ ID NO: 39 or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO: 148 or a modified sequence thereof; (4) the sense strand has the sequence shown in SEQ ID NO: 42 or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO: 151 or a modified sequence thereof; (5) the sense strand has the sequence shown in SEQ ID NO: 43 or a modified sequence thereof; and the antisense strand has the sequence shown in SEQ ID NO: 152 or a modified sequence thereof; (6) The sense strand has a sequence as shown in SEQ ID NO: 62 or a modified sequence thereof; and the antisense strand has a sequence as shown in SEQ ID NO: 171 or a modified sequence thereof.

5. A siRNA conjugate, characterized in that The conjugate comprises the siRNA duplex according to any one of claims 1 to 4, and a conjugated group connected thereto.

6. A nucleic acid-protein complex, characterized in that The nucleic acid-protein complex comprises the double-stranded region of the siRNA duplex according to any one of claims 1 to 4, and a nuclease; or, the nucleic acid-protein complex comprises the antisense strand of the siRNA duplex according to any one of claims 1 to 4, and a nuclease.

7. The nucleic acid-protein complex according to claim 6, wherein The nuclease is AGO protein.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the siRNA duplex according to any one of claims 1 to 4, the conjugate according to claim 5, or the nucleic acid-protein complex according to claim 6 or 7, and a pharmaceutically acceptable carrier.

9. Use of the siRNA duplex according to any one of claims 1 to 4, the conjugate according to claim 5, the nucleic acid-protein complex according to claim 6 or 7, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing or treating a disease associated with LPA gene expression.

10. Use of the siRNA duplex according to any one of claims 1 to 4, the conjugate according to claim 5, the nucleic acid-protein complex according to claim 6 or 7, or the pharmaceutical composition according to claim 8 in the preparation of a pharmaceutical composition for preventing or treating cardiovascular and cerebrovascular diseases.

11. Use of the siRNA duplex according to any one of claims 1 to 4, the conjugate according to claim 5, the nucleic acid-protein complex according to claim 6 or 7, or the pharmaceutical composition according to claim 8 in the preparation of a reagent and a kit for inhibiting LPA gene expression.

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